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Biomedical subjects

M M Frojmovic

Publications and source records attributed to M M Frojmovic.

At least 55 records · Page 3Linked to original sources
PubMed1987

Polyfunctional properties of hybridoma lupus anticoagulant antibodies.

Lupus anticoagulant antibodies are antiphospholipid antibodies which are characterized by their ability to prolong the clotting time in in vitro coagulation assays measuring the partial thromboplastin time (PTT). In our study, we have analyzed 11 hybridoma lupus anticoagulant antibodies and studied their reactivity with hexagonal and lamellar phospholipids, DNA, IgG, cytoskeletal components, and intact platelets. Our results demonstrate that hexagonal phase phospholipids but not lamellar phospholipids were able to neutralize the lupus anticoagulant activity of all 11 hybridoma antibodies. However, the lupus anticoagulant antibodies differed from one another in their reactivity with DNA, the Fc fragment of IgG, cytoskeletal proteins and platelets. Some of the lupus anticoagulant antibodies reacted with all of these structures, while others reacted with only some or none of the antigens tested. Isoelectric focusing gel analysis confirmed that this polyreactivity was due to a single antibody. Direct binding of lupus anticoagulant antibodies to intact platelets was shown to correlate with the DNA binding activity of the antibodies. Lupus anticoagulant antibodies were also capable of inducing a morphological shape change in platelets. Our data suggest that hybridoma lupus anticoagulant antibodies may react as lupus anticoagulant antibodies only, anti-DNA antibodies, rheumatoid factors, anticytoskeletal antibodies, and antiplatelet antibodies. The degree of overlap in these subgroups and the epitopes responsible for these multiple reactivities remain to be determined.

Antibodies, Antinuclear↗
PubMed1985

Platelet size and shape in hereditary giant platelet syndromes on blood smear and in suspension: evidence for two types of abnormalities.

Platelet size on blood smear is compared with platelet size and shape in suspension (i.e., whole blood and citrated platelet-rich plasma [PRP]) for normal donors and 16 patients with hereditary "giant" platelet syndromes (HGPS), including Bernard-Soulier syndrome (BSS) (seven patients), Montreal platelet syndrome (MPS) (three patients), May-Hegglin anomaly (one patient) and Rafael platelet defect (one patient). In whole blood platelet shape is normal for HGPS, but in PRP for 10 of 16 patients with HGPS there is a decrease in the proportion of smooth, discoid-shaped platelets (discocytes [D]). The platelets of all patients with HGPS had abnormally large mean volume (VT) and increased size on peripheral blood smear. Furthermore, 12 of 16 patients with HGPS, including six of seven donors with BSS, had abnormally large discocytes. The measured size of HGPS shape-changed platelets was compared with the size predicted from the size of the D by assuming that the relationship between the size of shape-changed platelets and D was the same as observed for normal donors. In this manner it was shown that for all donors with BSS and MPS, the shape-changed platelets are disproportionately larger than the D. In contrast, in the remaining patients with HGPS the size of the shape-changed platelets was consistent with the size predicted from the D. Examination of VT for MPS as a function of time after addition of 10 mumol/L adenosine diphosphate to PRP revealed an abnormal time course, thereby pointing to an abnormality in the mechanisms that regulate platelet size during shape change. With the lone exceptions of BSS and MPS, the size of platelets on blood smear was well correlated with the total platelet plasma membrane surface area as measured by the osmotic spherocyte method. Our observations point to two distinct abnormalities in platelet size in HGPS: a disproportion between the size of D and "shape-changed" platelets, which may be related to an abnormal shape change and which is observed only for MPS and BSS, and an abnormal increase in platelet size on blood smear, which appears to reflect the increased amount of platelet plasma membrane in other HGPS platelets.

Adenosine Diphosphate↗
PubMed1984

Reversal of acute experimental cerebral vasospasm by calcium antagonism with verapamil.

Acute vasospasm of the transclivally exposed basilar artery of anesthetised cats was produced by the subarachnoid injection of platelet-rich plasma (PRP) treated with enough adenosine diphosphate (ADP) to induce platelet aggregation and secretion. Vasorelaxation was produced by the topical application of the calcium antagonist verapamil. Changes in the internal diameter of the basilar artery were determined by measuring the blood column diameter from photomicrographs taken sequentially, at 5 minute intervals, through the operating microscope. Changes in blood vessel diameter are expressed as a plus or minus percentage of the pretreatment diameter. Arterial blood pressure and blood gas values were kept in the physiological range for the cat. The subarachnoid injection of PRP-ADP produced severe constriction of the basilar artery (mean constriction at 5 minutes after injection: -40.7% +/- 2.8 SEM). Platelet-free plasma, ADP alone and Elliott's A solution had no spasmogenic effect when injected into the subarachnoid space. The topical application of the calcium channel blocker verapamil (0.1 mg per kg) 30 minutes after the injection of PRP-ADP, with the basilar artery still in spasm (mean constriction: -23% +/- 3.5 SEM), produced prompt and dramatic vasodilation (mean dilation at 5 minutes after application: +52.7% +/- 18.1 SEM). This spasmolytic effect persisted in a decremental fashion for the 60 minute period of observation, by which time the previously constricted vessel had returned to its normal size. These observations indicate that the platelet fraction of whole blood may be involved in the genesis of acute vasospasm following subarachnoid hemmorrhage and that this phenomenon can be readily reversed by calcium antagonism.

Acute Disease↗
PubMed1984

Adrenaline and adenosine diphosphate-induced platelet aggregation require shape change. Importance of pseudopods.

The requirement of platelet shape change for early platelet aggregation measured from the disappearance of singlet platelets is compared for ADP and adrenaline. Platelet shape change is measured from pseudopod formation or from a change in main body volume or axial ratio. In the absence of shape change, platelets have a smooth discoid form and are referred to as discocytes. Citrated platelet-rich plasma prepared at 37 degrees C contains approximately 65% discocytes and approximately 35% discoid platelets, which possess pseudopods. The addition of ADP causes all platelets to change shape (pseudopods axial ratio and body volume) and 80% to 90% platelet aggregation. On the other hand, adrenaline causes only an approximately 15% increase in shape-changed platelets (pseudopods only) and approximately 33% platelet aggregation, in the presence of apyrase to minimize the effects of released ADP. Measurements of the shape of the nonaggregated platelets in the presence and absence of ongoing platelet aggregation induced by either ADP or adrenaline indicate that discocytes are not recruited into aggregates. For both ADP and adrenaline, the extent of platelet aggregation exceeds the extent of platelet shape change as measured from the disappearance of discocytes. However, in both cases, platelet aggregation does not exceed the total number of shape-changed platelets (i.e., pseudopods plus newly shape-changed discocytes), indicating that the pseudopods are also participating in platelet aggregation. Our results suggest that pseudopod formation is a common requirement for ADP- and adrenaline-induced platelet aggregation and is of greater importance than either changes in main body volume or axial ratio.

Adenosine Diphosphate↗
PubMed1984

Spontaneous platelet aggregation in a hereditary giant platelet syndrome (MPS).

The characteristics of spontaneous platelet aggregation (SPA) in a hereditary giant platelet syndrome (Montreal platelet syndrome, MPS) are examined. SPA was quantitated by microscopy from the decrease in single platelets in platelet-rich plasma (PRP). In contrast to normal donors, a significant proportion (20-50%) of platelets in MPS whole blood and PRP occurred in microaggregates typically containing 2-6 disk-shaped platelets. Stirring MPS-PRP at 1000 rpm for 10 minutes further increased the fraction of platelets in aggregates by 10-170%, the percentage increase not being correlated to the donor's platelet count (5000-220,000 microliters-1). Normal platelets resuspended in MPS platelet-poor plasma (PPP) did not undergo SPA, whereas MPS platelets resuspended in normal PPP or Ca2+-free, fibrinogen-free Tyrode's continued to show SPA. The increase in SPA could be inhibited by 10 microM prostaglandin (PG) E1, 150 mM ASA or glutaraldehyde or formaldehyde fixation; however, it was not inhibited by 10 nM PGI2 and was only partially inhibited by 1 microM 2-chloroadenosine and 1-10 units/ml apyrase. SPA in Acid-citrate-dextrose-PRP was much less than in PRP; however, SPA reoccurred on returning the platelets to platelet-free plasma or Tyrode's. Platelet aggregation (PA) could be increased over that due to SPA alone by the addition of adenosine diphosphate, adrenaline, collagen, ionophore A-23187, arachidonic acid and ristocetin, with results suggesting that the response to these agents is normal. The ristocetin-induced increase in PA was completely blocked by an IgG specific for Bernard-Soulier syndrome. In contrast, MPS platelets had a reduced sensitivity to thrombin, which appeared to be more pronounced at low platelet counts. There was no correlation between the thrombin insensitivity and the extent of SPA. Total adenosine triphosphate (ATP) and thrombin-induced release of ATP and platelet factor 4 appeared normal for MPS platelets. The ultrastructural features of MPS platelets were within normal limits except for an increased frequency of giant granules. SPA was observed for 5/5 MPS donors, but only one of three MPS donors' platelets evaluated for glycoprotein I and sialic acid content showed any measurable reduction as compared with normal controls. The above observations point to the existence of an as yet undetermined anomaly of MPS plasma membrane related to a fibrinogen and Ca2+ independent form of platelet aggregation.

Adenosine Triphosphate↗
PubMed1983

Sex-related differences in platelet morphology in whole blood (WB) and platelet-rich plasma (PRP).

The proportion of smooth, disc-shaped platelets (D) in freshly drawn, glutaraldehyde fixed whole blood (WB) and citrated platelet-rich plasma prepared at 37 degrees C is compared for male (N = 55) and female (N = 31) donors. Female donors have significantly more %D than male donors and the variability in %D measured on repeated occasions over a period of 3-69 months is less for female donors. WB and PRP gave similar results. The sex-related differences in %D were not related to hematocrit or to concomitant use of oral contraceptives. There were no significant sex-related differences in platelet mean volume or in platelet plasma membrane surface area as determined by the osmotic spherocyte method. However, the volume of D is smaller for male donors. It is concluded that the sex-related differences in platelet morphology do not represent intrinsic differences in platelet size or measurable total plasma membrane but represent a selective shape change activation of the larger D in the circulation of male donors. The significance of these observations for the sex-related differences in risk of cardiovascular disease and efficacy of anti-thrombotic therapy awaits appropriate prospective epidemiological studies.

Adolescent↗
PubMed1983

Turbidometric evaluations of platelet activation: relative contributions of measured shape change, volume, and early aggregation.

The relative contributions of ADP-induced changes in mean platelet axial ratio (R), mean volume (VT), pseudopod formation, refractive index, and early platelet aggregation to the changes in light transmission (%T) through stirred platelet suspensions (aggregometer) are determined. Measurement of the change in R within the first 10s of ADP addition indicates that the slope of the initial decrease in %T (5s) is in good agreement with that predicted from the theory of Latimer et al. (1977). However, when the changes in %T due to changes in VT are also considered, the observed rate of change of %T is shown to be 20-210% larger than predicted. These observations point to an additional process which produces a decrease in %T. It is shown that platelet aggregation occurring on this early time scale does not contribute to the initial changes in %T. Aggregation does lead to an increase in %T once there are greater than or equal to 30-40% singlet platelets aggregated which occurs at greater than or equal to 4-5s for 1-10 microM ADP. In the absence of aggregation, the extent of %T decrease at greater than 40s is greater than 25% smaller than predicted from the change in R and VT. It is suggested that at early times, changes in platelet refractive index produce significant decreases in %T which are on average offset by comparable increases due to the early volume increase, whereas at later times pseudopod formation has an important influence on %T.

Adenosine Diphosphate↗
PubMed1983

Physical, chemical and functional changes following platelet activation in normal and "giant" platelets.

Unactivated discocytes in healthy human donors have mean volumes of approximately 6.0 microns3 (range 3.8-7.5 microns3), while mean values for similarly-shaped discocytes obtained from donors with the hereditary "giant" platelet syndromes were either normal (one Bernard-Soulier syndrome (BSS) and all five members of a family with the Montreal platelet syndrome (MPS) or, on average, up to twice normal (range 6.4-13.8 microns3). This apparent heterogeneity is complicated by the much more consistent and significant observation that both BSS and MPS platelets undergo a defective hypervolumetric shape change following activation which is prolonged indefinitely, in contrast to a transient hypervolumetric change measureable in 1-5 s following ADP addition to normal platelets. It is suggested that the hypervolumetric shape change in both normal and "giant" platelets is accompanied by an increase in externalized plasma membrane surface area, with the most probable source being surface-connected canalicular system. Membrane glycoprotein I abnormalities were not detectable in platelets for 2/3 sibling MPS donors. The precise relation of these membrane changes to altered platelet functions is compared for normal and "giant" platelets, but largely remains to be experimentally determined. Early shape change appears tightly associated with early microscopically-measured aggregation (PA), with both PA and turbidimetrically-measured macroaggregation generally appearing normal to elevated for "giant" platelets.

Blood Coagulation Disorders↗
PubMed1983

Microscopic measurements of platelet aggregation reveal a low ADP-dependent process distinct from turbidometrically measured aggregation.

Platelet aggregation measured from the decrease in the number of nonaggregated platelets (PA) is compared with that measured from the increase in %T (TA) after ADP addition to a stirred platelet suspension. A number of properties distinguish PA from TA. Half-maximal extent of PA, measured at 3 or 10 sec after ADP addition is attained at significantly lower ADP concentrations ([ADP]) than for TA (velocity and extent), and in particular at [ADP] equal to or less than that required for shape change. PA appears minimally refractory to ADP under conditions causing maximal refractoriness of TA. Examination of the effect of three inhibitors of platelet aggregation acting on extracellular ionized calcium (EDTA) and on platelet adenylate cyclase (PGE1) and phosphodiesterase (anagrelide; BL-4162A) indicates that PA is generally less sensitive to these inhibitors than TA but, of greater significance, that PA and TA reveal aggregation processes with differing ADP sensitivities. It is suggested that these differences reflect different processes determining the strength of interplatelet interactions. Conventional aggregometry is insensitive for measuring the low ADP-dependent aggregation process.

Adenosine Diphosphate↗
PubMed1982

Platelet aggregation in laminar flow. I. Adenosine diphosphate concentration, time and shear rate dependence.

The relative contributions of physiologic activators and shear rates (G) on the formation and stability of platelet aggregates for in vitro laminar flow need to be characterized for conditions of low G believed to be important in vivo. Platelet aggregation was therefore studied in human citrated platelet-rich plasma (PRP) prepared at 37 degrees C for G = 0-190 sec-1 in laminar flow in a cone-in-plate device as a function of adenosine diphosphate (ADP) activation. Macroaggregation (TA) was assessed by visual observation and light transmission (%T), as routinely done in aggregometry, while actual percent aggregation (PA) was determined by microscopy of sub-samples from the decrease in single platelet concentration. Unactivated PRP never yielded shear-induced aggregation while samples pre-mixed with low [ADP] (less than or approximately 0.7 microM) led to large visible aggregate formation stable only for G less than or approximately 30-60 sec-1, apparently arising from pre-existing microaggregates. Direct ADP injections into the flow device showed that at low [ADP] (less than 1 microM), maximal values for both PA and TA were reached when step-wise increases in G reached less than or approximately 30 sec-1, with TA decreasing at higher G; these values were only some 10-20% of maximal values achieved with high (ADP] (greater than or approximately 4 microM), when PA was again maximal at G greater than or approximately 30 sec-1, but TA expressed as a rate of aggregation continued to increase with G up to 150 sec-1. The results are discussed in terms of the temporal shear-rate and ADP-dependent dynamics of a platelet stickiness factor, 's'.

Adenosine Diphosphate↗
PubMed1982

Platelet aggregation in laminar flow. II. Shear rate and ADP-dependent effects of acetylsalicylic acid and indomethacin.

The relative contributions of physiologic activators and shear rates (G) on the formation and stability of platelet aggregates arising from platelets treated with widely clinically used non-steroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) need to be characterized for conditions of low G believed to be important in vivo. In Part I of these studies it was found that platelets in human citrated platelet-rich plasma (PRP) undergo ADP-induced aggregation in laminar flow by apparently two distinct processes: a low ADP-dependent process, generating large visible aggregates stably only at low shear rates, and a higher ADP-threshold-dependent process yielding more complete and shear-resistant large aggregate formation. Similar studies of both macroaggregation (TA) and actual % aggregation (%PA) were made with ADP injections into PRP in the cone-in-plate device as a function of G and pre-treatments with ASA (200 microM) and indomethacin (10 microM). These drugs reduced %PA in part and yielded only reversible macro-aggregation for studies conducted at the higher [ADP] (4-6 microM) and at high G (greater than or approximately 120-150 sec-1), with no significant effects at low G (less than or approximately 20 sec-1). The results are discussed in terms of a platelet stickiness factor 's', ascribed to changes in fibrinogen binding sites reported for these drug actions, and related to in vivo flow and variable platelet activation conditions.

Adenosine Diphosphate↗
PubMed1981

Platelet adhesion to collagen is inhibited by adenosine diphosphate but unaffected by cell shape.

The effect of limited platelet activation, in the absence of aggregation, on the subsequent ability of rabbit platelets to adhere to collagen was studied in vitro. ADP at concentrations that initiate shape change ( greater than 0.01 micro M) reduce platelet adhesion. Shape change per se however, was not responsible since the time-dependence of the effect of ADP on shape change and adhesion is different and ADP induces reduced adhesion even when shape change is prevented with PGE or fixatives. Platelets shape-changed without ADP addition, e.g. by chilling or by low ethanol concentrations, display normal adhesion to collagen. It appears likely that upon binding to the platelet ADP induces a time-dependent alteration in the membrane, akin to refractoriness, that influences binding sites for collagen. The effect of ADP can be blocked by prior addition of AMP but not if the additions are reversed. The implications of the present finding for platelet adhesion studies are discussed.

Adenosine Diphosphate↗
PubMed1980

Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity.

The kinetics of ADP-induced human platelet shape change have been examined. Initial velocities of platelet shape change were estimated by two methods: (1) the slope of the initial decrease in light transmission through stirred, citrated platelet-rich plasma, and (2) direct examination of platelet morphologies by phase-contrast microscopy. In both cases, a value of the Hill coefficient, NH, significantly greater than 1 is obtained (2.0 +/- 0.2 and 1.8 +/- 0.2, respectively). The observed elevated value of NH is not due to a substantial fraction of the ADP being platelet bound, the presence of factors in the plasma, platelet heterogeneity, or the influence of the rate of platelet shape change reversion. Our observations suggest that ADP-induced platelet shape change may be a positively cooperative or "threshold" type response.

Adenosine Diphosphate↗
PubMed1979

Shape-changing agents produce abnormally large platelets in a hereditary "giant platelets syndrome (MPS)".

Geometries of platelets in citrated PRP obtained from normal donors (17) and donors (5) with a hereditary dominant giant platelet syndrome, herein referred to as "Montreal platelet syndrome" (MPS), are compared. The measured geometric axial ratio (rp = thickness/diameter) is used to classify platelet morphologies into three groups: discocytes (rp less than 0.5), disco-echinocytes (rp = 0.5 to 0.9), sphero-echinocytes (rp greater than 0.9). MPS discocytes are normal sized; however, MPS sphero-echinocytes and disco-echinocytes have mean volumes approximately two times larger than normal. It is demonstrated that these larger-than-normal sized MPS platelets can be produced directly from MPS discocytes by treatment with agents known to induce platelet shape change (adenosine diphosphate, thrombin, and incubation at 4 degrees C). Treatment of platelets obtained from normal donors which have been resuspended in MPS PPP and ADP or incubation at 4 degrees C causes the formation of normal-sized disco-echinocytes and sphero-echinocytes. The diameters of MPS disco-echinocytes are identical to the diameters of MPS platelets observed on peripheral blood smear, whereas those of MPS sphero-echinocytes are approximately 20% lower. It is suggested that the appearance of abnormally large platelets in MPS is related to a defect in the mechanism which regulates platelet size and shape during shape change.

Adult↗