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Biomedical subjects

R C Carroll

Publications and source records attributed to R C Carroll.

At least 55 records · Page 3Linked to original sources
PubMed1990

Effects of recombinant granulocyte colony-stimulating factor treatment on hematopoietic cycles and cellular defects associated with canine cyclic hematopoiesis.

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by 14-day cycles of neutropenia, monocytosis, thrombocytosis, and reticulocytosis. Platelets from CH dogs have decreased dense-granule serotonin pools and decreased aggregation responses to collagen, platelet-activating factor (PAF), and thrombin. Recombinant granulocyte colony-stimulating factor (rG-CSF) was administered (5 micrograms/kg, b.i.d.) to four CH and six normal dogs to determine if G-CSF therapy corrected qualitative platelet defects in CH dogs. Neutrophil counts increase to greater than 25,000 cells/microliters within 24 h after starting treatment in all dogs. Treatment with G-CSF blocked neutropenic episodes in the CH dogs. Platelet aggregation, and serotonin content and secretion were significantly (p less than 0.05) decreased in the CH dogs both before and during recombinant human (rh) G-CSF treatment compared to normal dogs. Neutrophil myeloperoxidase, a primary granule enzyme, was significantly (p less than 0.05) decreased in CH dogs and was not corrected by rhG-CSF treatment. Administration of rG-CSF to CH dogs eliminated cell cycles but apparently did not correct cellular defects in CH dogs. Identification of primary biochemical defects in cells from CH dogs may be crucial to investigating the biochemical basis for cyclic hematopoiesis.

Animals↗
PubMed1988

Regulation of fibrinolysis in aortic surgery.

The existence of inhibitors of plasminogen activator has been shown to play an important role in regulation of fibrinolysis and postoperative thrombosis. Platelets and endothelium are sources of plasminogen activator inhibitor (PAI). This study determines the contribution of platelet-released PAI to perioperative fibrinolytic shutdown. PAI levels were measured in 25 patients having aortic surgery. In nine patients the platelet-released PAI contribution was determined by in vitro activation of platelets with phorbol-myristate-acetate (PMA). Mean preoperative PAI levels (3.78 +/- 1.19 U/ml) were similar to controls (3.01 +/- 1.04 U/ml) (p greater than 0.05). Plasma PAI showed an operative increase to a maximum at 8 hours postoperatively and returning to preoperative values by the second postoperative day. In the nine patients who were subjected to studies with in vitro activation, the preoperative PAI level (4.0 +/- 0.9 U/ml) was elevated to 5.1 +/- 0.7 U/ml (p = 0.001) with PMA induction. Maximum stimulated release of platelet granule contents (platelet releasate) could account for an increase of only 1.0 U/ml compared with a postoperative increase of 2.3 U/ml. Postoperative mean peak plasma PAI (6.3 +/- 0.4 U/ml) could not be further elevated by induced release (6.3 +/- 0.4 U/ml) (p = 0.003). A statistically significant increase in PAI occurred in aortic surgery patients postoperatively. The platelet releasable pool of PAI contributed to the increase and was functionally exhausted postoperatively. Postoperative increases of PAI were twice that induced by platelet in vitro stimulation alone. The perioperative increase in PAI was partly due to platelet release.

Aorta↗
PubMed1986

Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets.

The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.

Blood Platelets↗
PubMed1986

The effect of tetradecanoylphorbol acetate on calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly induced by thrombin or arachidonate.

Tetradecanoylphorbol acetate (TPA) activates primarily only the protein kinase C pathway not the calcium ion-dependent pathway in platelets. The net effect of this split activation is that only the pseudopodal cytoskeleton assembles, not the contractile cytoskeleton needed for rapid secretion. In this study, platelets were first activated with TPA, then activated secondarily with either thrombin or arachidonate and the subsequent dense body secretion, calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly compared to these same processes in control platelets activated solely with either thrombin or arachidonate. Secretion was reduced as the length of time between the primary and secondary activation was increased; but at a 2-3 min interval, where the activation by TPA was essentially complete, the reduction in the total radiolabeled serotonin secreted was small. Furthermore, nearly normal cytosolic calcium-ion increases, phosphorylation of myosin light chain and contractile cytoskeletal development were induced by thrombin or arachidonate after this interval. Prior treatment of the platelets with 100 microM acetylsalicylate to block the cyclooxygenase-dependent pathway caused minor reduction in dense-body secretion induced by TPA or thrombin or the combination of both, but otherwise the relative results were comparable to the untreated platelets. Therefore, short-term prior activation of gel-filtered platelets with TPA, even at concentrations in excess of 100-times that required to saturate protein kinase C, does not prevent normal activation of the calcium ion dependent processes through either the cyclooxygenase-dependent or -independent pathway. Longer-term preincubations with TPA differentially inhibit the secretion response induced by thrombin and arachidonate.

Arachidonic Acid↗
PubMed1985

Reciprocal transmembranous receptor-cytoskeleton interactions in concanavalin A-activated platelets.

Concanavalin A (Con A) has been used to activate platelets, inducing a specific interaction between the glycoprotein IIb-IIIa complex and the cytoskeleton of the activated platelet. In agreement with this, we have shown that Con A activates human platelets, initiating phosphorylation, secretion, and cytoskeletal formation. Con A and cytochalasin B were used to demonstrate a reciprocal interaction of the glycoprotein complex with the platelet cytoskeleton. Additionally, we have shown that a similar reciprocity is provided by the multivalent fibrin-fibrinogen platelet interaction found in the thrombin-induced clot. Con A differs from other activators in precipitating an apparent cytoskeletal core despite a complete inhibition of platelet activation by prostaglandin E1. We suggest, from this result, that Con A may be cross-linking a membrane-associated cytoskeletal complex present in the unactivated platelet.

Actins↗
PubMed1984

The effects of lysophosphatidylcholine and related amphiphiles on platelet cytoskeletal assembly.

The effects of lysoPC, four other amphiphiles containing a linear 16 carbon alkane tail and chlorpromazine on platelet cytoskeletal assembly were compared. LysoPC and nonmetabolized amphiphiles all caused time-dependent inhibition followed by potentiation of thrombin-induced aggregation, serotonin secretion and cytoskeletal assembly in gel-filtered platelets, a result which ruled out hydrolysis of the amphiphiles as the mechanism of the time dependence. Hexadecanesulfonate was superior as a potentiator and cetyltrimethyl ammonium bromide (CTAB) was a better inhibitor. On the contrary, inhibition of platelet activation by arachidonate was not effected in a time-dependent manner and the actin-crosslinking proteins, actin-binding protein and myosin, were selectively prevented from incorporation into cytoskeletal cores, although protein phosphorylation and actin polymerization still occurred. Chlorpromazine also showed this selective inhibition of cytoskeletal assembly. LysoPC at concentrations which have been reported to cause development of filopodia did increase slightly the amount of actin present in Triton X-100-insoluble cores but not protein phosphorylation or incorporation of actin-crosslinking proteins. The effective concentrations of lysoPC and chlorpromazine can be predicted from the Meyer-Overton-Mullins rule of anesthesia which indicates their general effectiveness, but their specific effects only partially overlap.

Actins↗
PubMed1984

Multiple forms of phosphoinositide-specific phospholipase C of different relative molecular masses in animal tissues. Evidence for modification of the platelet enzyme by Ca2+-dependent proteinase.

The Mr distribution of phosphoinositide-specific phospholipase C in the supernatants isolated from a variety of animal tissues was analysed by high-performance gel-filtration chromatography. In most tissues, at least four peaks of activity were resolved. However, different tissues showed quite marked differences in the distribution of activity between these peaks. In rat heart, lung and kidney, the predominant form had Mr approx. 90000, whereas the predominant form in brain had Mr approx. 290000. In liver, the Mr-90000 form predominated, but this tissue also contained relatively large amounts of a form of Mr approx. 150000. Phospholipase C in these tissues from other animal species gave similar distributions of activity between the peaks. In supernatants prepared from platelets sonicated in the presence of leupeptin (0.5 mM) or EGTA (20 mM), the Mr-290000 form predominated. However, when leupeptin or EGTA (inhibitors of Ca2+-dependent proteinase) was omitted from the sonication buffer, the Mr-290000 form appeared to be replaced by a form of Mr 100000. Similar changes in Mr were not demonstrated with the other tissues. These results may be relevant to the intracellular regulation of phospholipase C, since Ca2+-dependent proteolysis has been reported to occur during platelet activation.

Animals↗
PubMed1984

Recycling of platelet phosphorylation and cytoskeletal assembly.

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.

Actins↗
PubMed1984

Retention of the glycoprotein IIb-IIIa complex in the isolated platelet cytoskeleton. Effects of separable assembly of platelet pseudopodal and contractile cytoskeletons.

To investigate the association of the putative platelet fibrinogen receptor (glycoprotein IIb-III(a) with the cytoskeleton, 125I-surface labeled human platelets washed by gel-filtration were activated under conditions which allow selective assembly of the platelet cytoskeleton. The four conditions were activation with arachidonate or phorbol 12-myristate 13-acetate (PMA) with and without pretreatment with cytochalasin E. Activation with arachidonate generates a complete cytoskeletal core (pseudopodal and contractile elements) while PMA activation forms only an actin plus actin-binding protein pseudopodal core. Pretreatment with cytochalasin E leads to actomyosin contractile core formation if arachidonate activated, and essentially blocks cytoskeletal development if PMA activated. Cytoskeletal cores from arachidonate or PMA-activated platelets retained 26 (+/- 3%) of the total 125I-IIIa. Pretreatment with cytochalasin E followed by arachidonate or PMA activation reduced the 125I-IIIa retention to near control levels (unactivated platelets: 4 +/- 2%). The role of aggregation vs. receptor occupancy in the retention of IIb-IIIa was assessed by activation of platelets with arachidonate in the presence of fibrinogen fragment D (0.6-12 mg/ml). Aggregation was blocked by increasing concentrations of fragment D reagent while cytoskeletal assembly was not altered. The IIIa retention correlated with extent of aggregation with maximal retention corresponding to full aggregation. To determine if cytoskeletal development is necessary for the expression of the fibrinogen binding site, binding studies were performed with unlabeled platelets and 125I-fibrinogen. The mean number of binding sites and the mean dissociation constant were not significantly different among the four activation conditions. Although the development of a platelet cytoskeletal core is not required for the expression of the fibrinogen binding site, the retention of the glycoprotein IIb-IIIa complex is dependent on fibrinogen-supported aggregation as well as the formation of the pseudopodal cytoskeleton.

Arachidonic Acid↗
PubMed1984

Effect of crosslinking on the structure of solubilized fibrin degradation products in whole plasma.

The purpose of these studies was to establish the validity of 125I fibrin autoradiography--SDS gel techniques for monitoring degradation products from whole plasma or blood clots. These methods can be used to study fibrin degradation not only in patients with congenital factor XIII deficiency, but also in patients with disseminated intravascular coagulation or deep vein thrombosis during the course of thrombolytic therapy. Such an assay might complement existing immunologic techniques to characterize fibrin degradation in vivo by providing an in vitro analysis of the rate and pattern of fibrin degradation in whole blood or plasma. Fibrin degradation was traced by Coomassie blue staining for protein and by autoradiography on SDS-PAGE of degradation products released from a 125I-labeled fibrin tracer. The degradation of non-crosslinked clots from purified fibrin supplemented with plasmin showed a typical release of X, Y, D, and E fibrin fragments. Subsequently, all X and Y fragments were digested to D and E fragments. The degradation of non-crosslinked washed clots prepared from plasma supplemented with plasmin reflected the same pattern. The degradation of non-crosslinked washed clots prepared from EDTA anticoagulated plasma without added plasmin also showed release of X, Y, D, and E fragments. However, in contrast to the non-crosslinked washed clots supplemented with plasmin, there was no additional degradation of the X and Y fragments. These studies established that the pattern of degradation of the 125I-radiolabeled fibrin tracer was similar to that of the total protein released from the fibrin clot as observed by protein staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
PubMed1983

Thrombomodulin blocks the ability of thrombin to activate platelets.

When thrombin is complexed to the endothelial cell surface receptor thrombomodulin, it loses its procoagulant activities in that it no longer clots fibrinogen or activates factor V. Studies were initiated to determine if complex formation also blocks thrombin's other major procoagulant function, the activation of platelets. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release [14C] serotonin. Binding studies using 125I-labeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. When thrombomodulin is added after thrombin has bound to the platelets, the thrombin rapidly redistributes onto the thrombomodulin. These data suggest that in addition to its other anticoagulant effects, thrombomodulin may also act to inhibit and/or reverse platelet activation by thrombin.

Animals↗
PubMed1982

31p nuclear magnetic resonance of platelet dense granule adenine nucleotides.

31P-NMR spectra of platelets were obtained after depletion of metabolic ATP and ADP by inhibitors. The remaining dense-granule nucleotides show prominent NMR peaks for rabbit, cow and pig, but not human, platelets. Each animal species has a unique and reproducible spectrum reflecting differences in dense-granule compositions, but in all, the dense-granule peaks are broader (by as much as 10-fold) than in spectra of solutions approximating the granule composition. Measurement of the transverse relaxation times, T2, for each resonance band yields intrinsic peak widths which generally are much narrower than observed peak widths. This result indicates that the broad dense-granule peaks are composed of intrinsically narrower peaks of slightly different chemical shifts, implying that the platelet dense granules of a given species are heterogeneous in composition. Our observations suggest that variations in divalent cation ratios and/or pH in the dense granules of a single animal can explain the spread of resonance positions.

Adenine Nucleotides↗
PubMed1982

Separable assembly of platelet pseudopodal and contractile cytoskeletons.

Unactivated platelets contain about 69% G actin and less than 10% of the contractile proteins in a cytoskeletal core resistant to extraction with 1% Triton X-100. Activation by thrombin leads, within 1 min, to the formation of pseudopodia and contractile gels, accompanied by the reduction of the G-actin content to about 22% and the development of cytoskeletal cores containing 70%-80% of the total actin and 60%-80% of the total myosin and actin-binding protein. Inhibition of pseudopodal formation by pretreatment with cytochalasin B before thrombin activation results in the loss of most of the actin-binding protein and about one third of the actin from the cytoskeletal core. Myosin incorporation and contractile gel formation are unaltered by this treatment. Conversely, activation with phorbol 12-myristate 13-acetate leads to pseudopodal but not contractile gel formation, with cytoskeletal cores containing mostly actin and actin-binding protein. These results demonstrate that there are separable cytoskeletal assembly processes in platelets for pseudopodal and contractile gel formation.

Actins↗
PubMed1982

Plasminogen, plasminogen activator, and platelets in the regulation of clot lysis. Reconstitution and patient studies.

Dilute clot lysis was assayed by release of soluble 125I fibrin degradation products from dPPP clots containing varying amounts of plasminogen activator and platelets. Plasminogen activator in the absence of platelets gave an approximately linear rate of lysis, with a rate proportional to its concentration. Addition of platelets to achieve normal clot retraction had little effect on the lysis rate in the absence of plasminogen activator or in the presence of very high plasminogen activator levels. However, with intermediate plasminogen activator levels, platelet-mediated clot retraction was associated with an accelerated rate of clot lysis when retraction reached 75% to 90%. The length of the lag phase before the start of the accelerated phase varied with the number of platelets and rate of clot retraction. The interaction of clot retraction and lysis was further explored in selected patients to determine (1) whether the contributions of platelet and plasma factors in these cases was similar to those seen in our studies of reconstituted plasma and (2) whether our experience with reconstituted systems could be used in the study of disorders of fibrinolysis involving platelets and fibrinolytic enzymes.

Blood Coagulation↗
PubMed1982

Phosphorylation of platelet actin-binding protein during platelet activation.

In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

Animals↗
PubMed1981

Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs.

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein in unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A2, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, though other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.

Arachidonic Acid↗
PubMed1981

Clot retraction facilitates clot lysis.

Platelet facilitation of clot lysis was studied using the dilute clot lysis assay, a standardized assay for fibrinolysis shown to correlate with the development of postoperative deep vein thrombosis. Clots prepared from dilute platelet poor plasma showed prolonged clot lysis when compared with clots prepared in a similar fashion from dilute platelet rich plasma. Since in the presence of platelets clot retraction or contraction occurred, we evaluated a possible direct contribution of retraction to clot lysis. Dilute platelet poor plasma clots were compacted by centrifugation, to a similar extent as that achieved during clot retraction in dilute platelet rich plasma. These clots now lysed at a rate that approached that seen with dilute platelet rich plasma clots. Using an alternate alternate approach, dilute platelet rich plasma clots were treated with cytochalasin B to prevent clot retraction. Such clots now showed prolonged lysis similar to that seen with dilute platelet poor plasma. The prolonged lysis of cytochalasin B treated dilute platelet rich plasma clots was corrected by artificial compaction of the clots. The results suggest that clot retraction markedly facilitates clot lysis, and shows that a major role of platelets to facilitate clot lysis is the effect of these cells to cause clot retraction.

Blood Platelets↗