Early assessment of the success of thrombolytic therapy by noninvasive markers.
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Biomarkers↗
Biomedical subjects
D R Abendschein
Publications and source records attributed to D R Abendschein.
Biochemical markers of myocardial injury. Is MB creatine kinase the choice for the 1990s?
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Animals↗
Importance of factor Xa in determining the procoagulant activity of whole-blood clots.
The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.
Animals↗
Inhibition of concomitant thrombin-mediated fibrin formation enhances clot lysis in whole blood.
To characterize potential mechanisms for enhanced thrombolysis in the presence of thrombin inhibitors, we measured lysis of 125I-fibrin-(ogen)- labelled clots after incubation in rotating tubes with whole blood containing tissue-type plasminogen activator (t-PA, 1500 ng/ml) and either saline or increasing concentrations of recombinant desulphatohirudin (hirudin), or heparin. Thrombin activity in washed clots was negligible, but incubation of clots with t-PA in non-anticoagulated blood resulted in marked thrombin activity within 5 min as measured by fibrinopeptide A generation. Incubation with hirudin over a range of concentrations increased clot lysis compared with t-PA alone (control) from 132 +/- 18% of control with 0.5 microgram/ml (n = 4) to 216 +/- 48% of control with 10 micrograms/ml (n = 4). Incubation with less than 0.5 U/ml of heparin attenuated clot lysis (35 +/- 22% of control with 0.08 U/ml, n = 3) while concentrations > or = 0.5 U/ml increased lysis (178 +/- 13% of control with 1.7 U/ml, n = 4). Similar results were obtained for incubations in recalcified platelet-poor plasma indicating that platelets are not required for the enhancement of clot lysis induced by thrombin inhibitors. However, incubations in recalcified plasma from a patient with afibrinogenaemia abolished the increased lysis observed with 10 micrograms/ml of hirudin and addition of physiological concentrations of fibrin monomer (400-800 nM) or fibrinogen degradation products (500 nM) to the mixture of whole blood, t-PA and hirudin blunted the extent of clot lysis. Thus, inhibition of thrombin activity induced by exposure of clots to t-PA prevents concomitant formation of fibrin that attenuates fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood↗
Nature and time course of generation of isoforms of creatine kinase, MB fraction in vivo.
OBJECTIVES: This study was designed to characterize the nature and time course of carboxy-terminal lysine cleavages from the tissue isoform of MB creatine kinase (CK) in vivo. BACKGROUND: Rapid conversion of the tissue isoform of MM CK to two additional circulating isoforms with one or both carboxy-terminal lysines cleaved facilitates early detection of new tissue isoform release after acute myocardial infarction and coronary recanalization. Characterization of changes in plasma MB CK isoform profiles, potentially enhancing specificity even further, has been hindered by difficulties in separating the isoform products and elucidation of carboxy-terminal lysine cleavages underlying their formation. METHODS: Isoform species with carboxy-terminal lysine present on B-monomers were separated from those from which lysine had been cleaved by anion exchange chromatography. Carboxy-terminal lysine on M-monomers was assayed with the use of a monospecific antibody. RESULTS: MB CK in four pooled plasma samples from among 77 normal subjects exhibited carboxy-terminal lysine on 48 +/- 21% (mean +/- SEM) of B-monomers and 82 +/- 12% of M-monomers. Within the 1st 16 h after the onset of acute myocardial infarction, virtually all M- and B-monomers exhibited carboxy-terminal lysine, indicating release into plasma and the lack of rapid cleavage of lysine from the tissue isoform. After 20 to 30 h, 43 +/- 9% (three pools from 19 patients) of B-monomers and 95 +/- 10% of M-monomers exhibited lysine at the carboxyl terminus. After 40 to 50 h, 13 +/- 13% (four pools from 34 patients) of B-monomers and 46 +/- 19% of M-monomers still retained carboxy-terminal lysine. CONCLUSIONS: In contrast to MM CK, the tissue isoform of MB CK undergoes slow cleavage of lysine from both monomers in vivo. Sequential cleavage of lysine first from the carboxyl terminus of B-monomers and subsequently from M-monomers is consistent with generation of at least two additional isoforms. Development of assays capable of resolving all of the isoforms of MB CK that can occur in vivo might increase sensitivity for early detection of new tissue isoform release associated with acute myocardial infarction and coronary recanalization compared with currently available assays that resolve only two species.
Chromatography, Ion Exchange↗
Augmentation of plasminogen activator inhibitor type 1 activity in plasma by thrombosis and by thrombolysis.
Both activation of platelets and elevation of plasminogen activator inhibitor type 1 (PAI-1) activity in plasma have been associated with acute myocardial infarction. Growth factors from platelet alpha-granules have been shown to increase PAI-1 synthesis in liver and endothelial cells in culture. The present study was designed to determine whether activation of platelets in vivo increases PAI-1 activity in plasma, thereby potentially attenuating thrombolysis. Carotid arteries in rabbits were stimulated with transluminal anodal current to initiate thrombosis manifested initially by cyclic flow variations known to reflect platelet activation. Flow was monitored with Doppler flow probes. Plasma PAI-1 activity (mean +/- SEM) assayed spectrophotometrically increased from 6.8 +/- 0.8 arbitrary units (AU)/ml to a peak of 19.1 +/- 2.9 AU/ml (n = 15) 4.8 +/- 0.6 h after the onset of cyclic flow variations. The magnitude of peak PAI-1 values correlated closely with the frequency and duration of antecedent cyclic flow variations. Complete thrombotic occlusion did not elevate PAI-1 beyond that seen with severe, repetitive partial occlusions (18.7 +/- 4.6 vs. 19.6 +/- 3.8 AU/ml). However, when recanalization of completely occluded vessels was induced with tissue-type plasminogen activator (t-PA), plasma PAI-1 increased more markedly (from 5.6 +/- 0.7 to 112.8 +/- 22.3 AU/ml, n = 11), exceeding the increase after corresponding intervals in animals in which t-PA failed to induce recanalization (from 5.2 +/- 1.1 to 28.3 +/- 6.1 AU/ml, n = 6). Thus, activation of platelets accompanying thrombosis or thrombolysis, or both, markedly increases PAI-1 activity in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Inhibition of cyclic flow variations and reocclusion after thrombolysis in dogs by a novel antagonist of platelet-activating factor.
To determine whether platelet-activating factor is a specific mediator of cyclic flow variations in damaged stenotic arteries and whether it contributes to reocclusion after thrombolysis, femoral arteries in anesthetized dogs were subjected to mural injury and high grade stenosis to induce cyclic flow variations (28 +/- 4/h) or methods selected to elicit platelet-rich and fibrin-rich thrombosis. Oral administration of a novel triazolobenzodiazepine (U46,195 [10 mg/kg]) that selectively inhibits platelet-activating factor abolished cyclic flow variations within 120 min and for greater than or equal to 2 h thereafter compared with persistent flow variations in dogs given saline solution. Platelet aggregation induced ex vivo with platelet-activating factor was inhibited in parallel with in vivo inhibition of cyclic flow variations after administration of U46,195. However, buccal mucosa bleeding time was not affected. After thrombosis, administration of U46,195 before thrombolysis was induced with human recombinant tissue-type plasminogen activator (1.7 mg/kg intravenously over 60 min) prevented reocclusion within 120 min in six of eight and six of seven arteries by platelet-rich and fibrin-rich thrombosis, respectively. In contrast, in dogs given saline solution, reocclusion occurred in eight of eight (p = 0.007 compared with U46,195) and five of eight arteries by platelet-rich and fibrin-rich thrombosis, respectively. Thus, both cyclic flow variations and reocclusion after thrombolysis appear to be mediated in part by platelet-activating factor. The results suggest that inhibition of platelet-activating factor with specific antagonists may be useful in reducing platelet-mediated occlusion of coronary arteries without eliciting bleeding.
Administration, Oral↗
Relative efficacy of antithrombin compared with antiplatelet agents in accelerating coronary thrombolysis and preventing early reocclusion.
BACKGROUND: Optimal coronary thrombolysis should be prompt and persistent. Although activation of platelets and increased thrombin activity have been associated with clinical thrombolysis, the role of each in delaying thrombolysis or inducing early coronary reocclusion has been difficult to define. METHODS AND RESULTS: In conscious dogs with coronary thrombosis induced by electrical current, we assessed the impact on the rapidity of thrombolysis and the incidence of reocclusion of two types of adjunctive treatment given concomitantly with intravenous tissue-type plasminogen activator (t-PA): 1) inhibition of platelet function with a peptide mimetic antagonist of platelet glycoprotein IIb/IIIa receptors or with lysine acetylsalicylic acid (ASA) and 2) inhibition of thrombin activity with recombinant hirudin or with heparin. ASA but not the receptor antagonist shortened the time to thrombolysis with t-PA (20 +/- 13 [mean +/- SD] minutes with ASA, 36 +/- 15 minutes with receptor antagonist, and 43 +/- 16 minutes with the saline control). Reocclusion occurred promptly after completion of the infusion of t-PA in all seven dogs given saline. Reocclusion was delayed and prevented in some dogs within 90 minutes after the end of the infusion of t-PA by both antiplatelet agents but still occurred in 42% despite continued inhibition of platelet function (i.e., three of six dogs given ASA and two of six given receptor antagonist). In contrast, inhibition of thrombin activity with recombinant hirudin in a dose that prolonged the partial thromboplastin time modestly (1.5-2-fold) resulted in accelerated lysis (19 +/- 10 minutes) and prevention of reocclusion in each of six dogs. Heparin given in doses that elicited similar prolongation of the partial thromboplastin time did not accelerate lysis nor prevent reocclusion, which occurred in five of six dogs. CONCLUSIONS: Inhibition of thrombin by recombinant hirudin facilitates thrombolysis and maintains patency of coronary arteries recanalized with t-PA particularly effectively. The benefit conferred may reflect direct anticoagulant effects plus diminished activation of platelets secondary to decreased thrombin activity.
Animals↗
Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor.
BACKGROUND: This study was designed to determine whether arterial reocclusion after thrombolysis can be prevented by lipoprotein-associated coagulation inhibitor (LACI), a physiological inhibitor of tissue factor-induced coagulation mediated by the extrinsic pathway. METHODS AND RESULTS: Thrombosis was induced in femoral arteries of anesthetized dogs with the use of anodal current to elicit extensive vascular injury and formation of platelet-rich thrombi in one artery and with thrombogenic copper wire to elicit fibrin-rich thrombi without appreciable vascular injury in the contralateral artery. Recanalization of both vessels was induced with t-PA (1.7 mg/kg i.v. over 1 hour) and verified with Doppler flow probes. Reocclusion occurred within 2 hours in seven of seven arteries with electrical injury-induced thrombosis and in four of seven arteries with copper wire-induced thrombosis in the absence of LACI. In dogs given infusions of recombinant DNA-produced LACI (225 micrograms/kg over 15 minutes, followed by 4 micrograms/kg/min i.v.) after completion of the infusion of t-PA, no reocclusion occurred during the 2-hour interval of observation in any of the five arteries subjected to electrical injury (p less than 0.001), and cyclic partial occlusions were nearly abolished (0.4 +/- 0.4/hr in LACI-treated dogs compared with 13.7 +/- 5.5/hr in saline-treated dogs, p less than 0.0001). In contrast, reocclusion occurred in two of five arteries with indwelling copper wires, and cyclic partial occlusions were unaffected despite LACI. LACI prolonged the partial thromboplastin time modestly (1.7 +/- 0.2 x baseline) but did not affect platelet counts or aggregation assessed ex vivo. CONCLUSIONS: Inhibition of the extrinsic pathway of coagulation with LACI prevents thrombotic arterial reocclusion after thrombolysis in vessels subjected to extensive vascular injury. Our results demonstrate that activation of the extrinsic pathway plays a critical role in thrombotic reocclusion and that LACI provides a highly targeted approach to facilitate sustained recanalization without directly inhibiting platelets.
Animals↗
Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.
Pressure overload of the left ventricle induces synthesis of creatine kinase isoenzymes. To determine whether this response is associated with an altered pattern of creatine kinase gene expression, we induced arterial hypertension in rats by suprarenal aortic banding. After 4 days, left ventricular myocardium from hypertensive (n = 7) and normotensive, sham-operated (n = 5) rats was analyzed for isoenzyme activities by chromatography; M and B creatine kinase subunit protein by Western blot; and M, B, and mitochondrial creatine kinase mRNA by Northern blot. Although total creatine kinase activity increased in hypertensive (1,096 +/- 214 IU/g left ventricle) compared with normotensive rats (648 +/- 81 IU/g left ventricle, p less than 0.01), the relative proportions of the cytoplasmic and mitochondrial isoenzymes did not change. The mass of M and B subunits increased 1.9- and 2.7-fold, respectively, in hypertensive compared with control rats. Similarly, the mRNA for M and B subunits as well as mitochondrial creatine kinase increased 2.6-, 1.6-, and 1.8-fold, respectively, in hypertensive rats compared with control rats. Thus, increased energy requirements in acute pressure overload are met by generalized induction of creatine kinase mRNA and subunit protein and not by an isoenzyme switch.
Animals↗
Rapid diagnosis of myocardial infarction and reperfusion by assay of plasma isoforms of creatine kinase isoenzymes.
Early, reliable detection of acute myocardial infarction and of coronary artery recanalization, in patients receiving thrombolytic agents, is essential to guide the course of therapy. Because the MM and MB isoenzymes of creatine kinase (CK) released from myocardium, undergo time-dependent removal of carboxyl terminal lysine residues from each monomer during exposure to circulating carboxypeptidase N, plasma profiles of the resulting isoforms are altered promptly and markedly after the release of new tissue isoenzymes. This paper reviews the results of experimental and preliminary clinical studies, showing the potential for rapid diagnosis of myocardial infarction and coronary artery recanalization by analysis of isoforms of CK isoenzymes in plasma.
Clinical Enzyme Tests↗
Optimized preservation of isoforms of creatine kinase MM isoenzyme in plasma specimens and their rapid quantification by semi-automated chromatofocusing.
We report a convenient chromatofocusing procedure for rapid and sensitive quantification of isoforms of the MM isoenzyme of creatine kinase (EC 2.7.3.2) in plasma and efficient methods for preserving isoform profiles during handling of specimens. The assay involves use of prepacked, re-usable Mono P chromatofocusing columns and a "Fast Protein Liquid Chromatograph" (FPLC) system with on-line detection of isoform enzymatic activity in column effluent. Profiles of isoforms are analyzed within 25 min with the use of a 1-mL column; the lower limit of sensitivity for CK activity is 5 mU, and recovery of each isoform is within 1% of the amount added to plasma. Collection of blood specimens in Vacutainer Tubes containing 28.5 mumol of EDTA (final concentration in plasma, 7 to 10 mmol/L) inhibited carboxypeptidase activity in plasma by 76%, sufficient to essentially abolish isoform conversion in vitro at room temperature. These methods should facilitate applications of isoform analysis for diagnosis of myocardial infarction and coronary artery recanalization.
Autoanalysis↗
Deaggregation of human platelets in vitro by an RGD analog antagonist of platelet glycoprotein IIb/IIIa receptors.
Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa receptors is essential for normal platelet aggregation. We investigated whether inhibition of GP IIb/IIIa receptors with arginine-glycine-aspartate-O-methyltyrosine amide (RGDY), an analog of the receptor recognition sequence found in fibrinogen, is associated with platelet deaggregation. Platelets from five healthy human subjects that were maximally aggregated by addition of adenosine diphosphate to platelet-rich plasma were deaggregated in a dose-dependent manner by subsequent addition of RGDY (86.5 +/- 6.2%, 68.2 +/- 4.3%, 44.9 +/- 6.4%, and 31.6 +/- 4.1% for RGDY concentrations of 400, 200, 133, and 67 microM, respectively vs. 7.8 +/- 2.9% for saline control samples, p less than 0.0001). The extent of deaggregation was decreased as the time of addition of RGDY (400 microM) after maximal aggregation increased (85.6 +/- 6.7%, 58.5 +/- 12.6%, 47.1 +/- 2.7%, and 37.1 +/- 4.2% for 0, 1, 3, and 5 minute intervals, respectively, vs. 8.3 +/- 3.1% in control samples, n = 4, p less than 0.0001) consistent with the occurrence of irreversible aggregation. Thus, RGDY can rapidly and extensively deaggregate human platelets under certain conditions which may enhance its antithrombotic efficacy.
Adult↗
Transmural gradients of coronary flow reserve with physiologically and morphometrically defined stenoses in dogs.
Coronary angiography permits identification of stenotic lesions but underestimates their severity and does not provide information regarding their physiologic significance. Evaluation of coronary flow reserve by means of selective coronary artery Doppler flow catheters or quantitative arteriography has been proposed to obtain this information. However, these techniques may not accurately reflect transmural gradients in flow. We evaluated the relationship between flow reserve defined with an epicardial Doppler flow probe and the transmural gradient of flow measured with radiolabeled microspheres in 21 dogs with graded stenoses and correlated results with coronary artery geometry measured morphometrically. Four groups of dogs were studied. In five control dogs without stenosis, reactive hyperemia after 20 seconds of complete coronary occlusion was 4.5 +/- 1.5 (mean +/- SD) times resting flow with an endocardial/epicardial flow ratio at peak flow of 1.0 +/- 0.2. When reactive hyperemia was blunted (without affecting resting flow) by 50% (n = 6), 75% (n = 5), or was abolished completely (n = 5) by coronary stenosis, the endocardial/epicardial flow ratio at peak flow was 1.0 +/- 0.3, 0.7 +/- 0.2, and 0.5 +/- 0.1, respectively. Cross-sectional area of the stenosed segment was reduced by 85.6 +/- 3.5%, 91.1 +/- 2.2%, and 92.8 +/- 4.3% in these groups, respectively. Thus in dogs with stenoses exceeding 86% of the cross-sectional area, endocardial flow reserve is compromised disproportionately compared with epicardial flow reserve, suggesting that clinical measurements of coronary flow reserve may underestimate the physiologic significance of coronary stenoses.
Animals↗
Enzymatic estimation of the extent of irreversible myocardial injury early after reperfusion.
To determine whether the extent of infarction can be estimated enzymatically soon after reperfusion, the rate of increase of creatine kinase (CK) activity in plasma early after coronary recanalization was compared with infarct size in 18 dogs and 10 patients. In dogs, reperfusion was initiated 2 to 4 hours after coronary occlusion. CK activity was measured in serial plasma samples and infarct size was assessed histochemically at 24 hours. A substantial and consistent fraction of the total CK appearing in plasma over 24 hours (cumulative CK) appeared in plasma soon after reperfusion, i.e., 21 +/- 2% (SE) within 30 minutes and 38 +/- 3% within 1 hour. The rate of increase of plasma CK activity correlated closely with infarct size when CK release was measured during the first 30 minutes (r = 0.92) or 60 minutes (r = 0.92) after reperfusion (n = 18). Similarly, in patients the rate of increase of CK activity measured within 2.5 hours of the time of reperfusion was closely related to infarct size delineated by positron emission tomography 1 to 2 weeks later (r = 0.93). Thus the rate of appearance of CK in plasma early after reperfusion reflects the extent of irreversible injury ultimately sustained and provides a criterion likely to be useful for prospective identification of patients at high risk after coronary recanalization.
Adult↗
Optimal criteria for rapid detection of myocardial reperfusion by creatine kinase MM isoforms in the presence of residual high grade coronary stenosis.
Analysis of isoforms of MM creatine kinase (CK) in plasma is being developed as a means for rapid detection of coronary recanalization in patients given thrombolytic agents. To determine whether flow-limiting residual stenosis typical of that seen in patients affects plasma isoform profiles, stenosis sufficient to preclude reactive hyperemia was induced in dogs before coronary occlusion, followed by recanalization in 2 h. Plasma activities of the MM CK isoform released from myocardium (MM3) and its two conversion products elaborated sequentially (MM2 and MM1) were assayed in serial samples with a rapid quantitative chromatofocusing procedure. Reperfusion in 10 dogs shortened the mean intervals (+/-SD) to the occurrence of peak MM3 activity (3.7 +/- 0.9 h), peak MM3 expressed as a percent of total CK activity (MM3%, 2.5 +/- 0.3 h) and the maximal ratio of MM3 to MM1 (2.7 +/- 0.3 h) compared with results in 10 control dogs without reperfusion. Nevertheless, the appearance of these peaks was delayed by 8% to 57% when residual stenosis was present. In contrast, the rate of increase of MM3% was delineated before the peak, was fivefold greater with recanalization (1.19 +/- 0.46 versus 0.26 +/- 0.11% min-1 in control dogs) and was not attenuated by residual stenosis. Thus, this criterion appears capable of delineating recanalization early after thrombolysis whether or not high grade residual stenosis is present.
Animals↗
Prevention of reoccluding platelet-rich thrombi in canine femoral arteries with a novel peptide antagonist of platelet glycoprotein IIb/IIIa receptors.
The composition of an evolving arterial thrombus may be a determinant of how effectively pharmacologic agents prevent reocclusion after initially successful thrombolysis. In this study, reoccluding platelet- or fibrin-rich thrombi as delineated by scanning electron microscopy were produced selectively in the femoral arteries of dogs with the use of electrically induced vascular injury or implantation of copper wire, respectively. Initial thrombolysis after intravenous infusion of tissue-type plasminogen activator (1 mg/kg over 30 minutes) was less frequent in the preparation producing platelet-rich thrombi than in that producing fibrin-rich thrombi (lysis in 19 of 24 versus 18 of 18, p = 0.06). In dogs with initial arterial recanalization, intravenous infusion of arginine-glycine-aspartate-O-methyltyrosine amide (RGDY), which competes with fibrinogen for binding to platelet glycoprotein IIb/IIIa receptors, prevented reocclusion caused by recurrence of platelet-rich thrombi in six of six dogs within 90 minutes; reocclusion occurred in five of seven saline-infused control dogs (p = 0.02). RGDY was only partially effective in preventing reocclusion caused by recurrence of fibrin-rich thrombi (reocclusion in three of six versus five of six controls, p = 0.54). Similar results were obtained with aspirin in both preparations. At least 98% of platelet aggregation induced ex vivo by collagen was inhibited by either RGDY or aspirin. In contrast with aspirin, however, platelet function returned to normal within 1 hour after discontinuation of RGDY. Thus, the relative proportions of platelets or fibrin incorporated into thrombi influence the efficacy of both tissue-type plasminogen activator for inducing thrombolysis and antiplatelet agents for preventing reocclusion. RGDY is a potent, short-acting inhibitor of platelet aggregation that effectively prevents reocclusion under conditions in which platelet deposition predominates.
Amino Acid Sequence↗
Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.
Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.
Amino Acid Sequence↗