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PubMed1999

Decreased platelet serotonin transporter sites and increased platelet inositol triphosphate levels in patients with unipolar depression: effects of clomipramine and fluoxetine.

BACKGROUND: The central serotonergic system has been implicated in the pathophysiology of depression and in the mechanism of the action of antidepressant drugs. The human platelet has been proposed as a peripheral model of central serotonergic neurons. METHODS: Six peripheral serotonergic parameters were determined simultaneously in 27 patients with unipolar depression before and after 2, 4, and 12 weeks of clomipramine or fluoxetine treatment according to the psychiatrist. RESULTS: In patients with depression versus matched control subjects, platelet [3H]paroxetine binding sites were found to be significantly decreased (2.10 +/- 0.70 versus 3.88 +/- 0.77 fmol/10(9) platelets; P = .0001), platelet serotonin (5-HT) content was found to be significantly decreased (1.90 +/- 1.52 versus 2.74 +/- 1.12 nmol/10(9) platelets; P = .001), and platelet inositol triphosphate levels were found to be significantly increased (2.85 +/- 0.70 versus 1.85 +/- 0.77 fmol/10(9) platelets; P = .0001). No significant difference between patients and control subjects was found for platelet [3H]-lysergic acid diethylamide ([3H]LSD) binding sites, aggregation tests with 5-HT or adenosine diphosphate and plasma 5-HT levels. Treatment with both clomipramine and fluoxetine gradually further reduced the density of platelet [3H]paroxetine binding sites and induced a dramatic decrease in platelet and plasma 5-HT levels. With clomipramine, the decreased blood 5-HT levels are associated with increased platelet [3H]LSD binding sites and aggregation responses. After 12 weeks, nonresponders to both treatments had platelet inositol triphosphate levels that were still increased (2.81 +/- 0.75 fmol/10(9) platelets) when responders levels were not different from those of control subjects (1.41 +/- 0.45 versus 1.70 +/- 0.25 fmol/10(9) platelets). CONCLUSIONS: Drug-free patients with depression had simultaneously decreased 5-HT transporter (5-HTT) sites and overstimulated phosphoinositide signaling systems. Clomipramine and fluoxetine treatments, which further decreased the density of 5-HTT sites, allowed platelet inositol triphosphate levels to return to normal values only in responders.

Adult↗
PubMed1981

Mean platelet volume. The inverse relation of platelet size and count in normal subjects, and an artifact of other particles.

We measured whole-blood mean platelet volume (MPV) and platelet count, determined by the Coulter Counter model S-Plus, in 683 normal subjects. There was nonlinear, inverse relation between MPV and platelet count throughout the normal range of platelet count: the change in MPV was most pronounced at the lower platelet counts. Because of this non-linear relation, "platelet-crit," the product of platelet count and MPV, was not constant, but varied directly with platelet count. Thus, the definition of "normal" values for MPV requires simultaneous reference to platelet count. In the whole blood of 13 patients, non-platelet cell fragments spuriously increased the automated platelet count. Seven patients with erythrocyte fragmentation had spuriously high MPV also: correct MPV and platelet count could be obtained from platelet rich plasma (PRP). In five patients with leukemic blast fragmentation, MPV remained spuriously low and platelet count spuriously high in PRP: only phase count allowed correct count. A patient with megakaryoblastic leukemia had megakaryoblast fragments confused for platelets.

Adolescent↗
PubMed2002

Anti-platelet effects of GPIIb/IIIa and P-selectin antagonism, platelet activation, and binding to neutrophils.

Platelet activation with GPIIb/IIIa binding to fibrinogen, aggregation and interaction with leukocytes constitute the principal mediator of thrombosis. Although the clinical benefits of GPIIb/IIIa antagonists have been documented, the relationship between their anti-platelet properties, platelet activation and binding to leukocytes is still debated. We investigated the effects of abciximab, tirofiban, roxifiban, and an anti-P-selectin blocking monoclonal antibody (Mab) on isolated human platelet aggregation using optical aggregometer, and on platelet P-selectin and GPIIb/IIIa expression, and platelet-neutrophil binding using flow cytometry. Thrombin at 0.025 U/ml induced maximal platelet aggregation (76.3 +/- 2.6%), P-selectin expression (88.5 +/- 4%), GPIIb/IIIa activation (PAC-1 binding, 86.2 +/- 8.9%) and platelet-neutrophil binding (58.0 +/- 6.4%). The GPIIb/IIIa antagonists inhibited in a concentration-dependent manner platelet aggregation (IC50 of 100 nM for abciximab and tirofiban and 50 nM for roxifiban) and PAC-1 binding, without any effect on P-selectin. None of these agents affected significantly platelet-neutrophil binding, whereas an anti-P-selectin Mab abolished this binding and amplified the effect of abciximab on platelet aggregation. These results indicate that the effects of these GPIIb/IIIa antagonists on platelet aggregation are not related to inhibition of platelet activation, as P-selectin levels and platelet-neutrophil binding remained unaffected, and highlight the participation of P-selectin with GPIIb/IIIa in platelet aggregation.

Abciximab↗
PubMed1988

Splenic platelet kinetics and platelet production after major reconstructive vascular surgery.

By using 111In-labelled platelets and dynamic gamma camera scintigraphy, platelet production rate and intrasplenic platelet kinetics were determined in 13 patients at 1 and 4 months after aortic reconstructive vascular surgery with implantation of dacron prostheses. A significant decrease in platelet production rate and venous platelet count was recorded over time after surgery. Irrespective of whether the exchangeable splenic platelet pool was estimated from initial recovery of platelet-bound radioactivity or from compartmental analysis, the size of this pool was significantly lower at the first study; a change in intrasplenic platelet transit time accounted for the observed difference. Platelet mean life-span increased over time after surgery but the difference between the duplicate studies was not statistically significant. It can be concluded that there is a reduction of platelet production rate and venous platelet count over time after major reconstructive vascular surgery. The early postoperative elevation in the platelet count is mainly the result of an increased platelet production and to a lesser degree due to redistribution of platelets between the splenic platelet pool and general circulation.

Aged↗
PubMed1997

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Annexin A5↗
PubMed1998

Erythrocyte promotion of platelet reactivity decreases the effectiveness of aspirin as an antithrombotic therapeutic modality: the effect of low-dose aspirin is less than optimal in patients with vascular disease due to prothrombotic effects of erythrocytes on platelet reactivity.

BACKGROUND: Aspirin (acetylsalicylic acid, ASA) is widely used for secondary prevention of ischemic vascular events, although its protection only occurs in 25% of patients. We previously demonstrated that platelet reactivity is enhanced by a prothrombotic effect of erythrocytes in a thromboxane-independent manner. This diminishes the antithrombotic therapeutic potential of ASA. Recent data from our laboratory indicate that the prothrombotic effect of erythrocytes also contains an ASA-sensitive component. In accordance with this observation, intermittent treatment with high-dose ASA reduced the prothrombotic effects of erythrocytes ex vivo in healthy volunteers. In the present study, the effects of platelet-erythrocyte interactions were evaluated ex vivo in 82 patients with vascular disease: 62 patients with ischemic heart disease treated with 200 mg ASA/d and 20 patients with ischemic stroke treated with 300 mg ASA/d. METHODS AND RESULTS: Platelet activation (release reaction) and platelet recruitment (fluid-phase proaggregatory activity of cell-free releasates from activated platelets) were assessed after collagen stimulation (1 microg/mL) of platelets, platelet-erythrocyte mixtures, or whole blood. Platelet thromboxane A2 synthesis was inhibited by >94% by ASA administration in all patients. Importantly, platelet recruitment followed one of three distinct patterns. In group A (n=32; 39%), platelet recruitment was blocked by ASA both in the presence and absence of erythrocytes. In group B (n=37; 45%), recruitment was abolished when platelets were evaluated alone but continued in the presence of erythrocytes, indicating a suboptimal effect of ASA on erythrocytes of this patient group. In group C (n= 13; 16%), detectable recruitment in stimulated platelets alone persisted and was markedly enhanced by the presence of erythrocytes. CONCLUSIONS: In two thirds of a group of patients with vascular disease, 200 to 300 mg ASA was insufficient to block platelet reactivity in the presence of erythrocytes despite abolishing thromboxane A2 synthesis. Platelet activation in the presence of erythrocytes can induce the release reaction and generate biologically active products that recruit additional platelets into a developing thrombus. Insufficient blockade of this proaggregatory property of erythrocytes can lead to development of additional ischemic complications.

Adult↗
PubMed2005

Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia.

Interaction between target cell CD47 and the inhibitory macrophage receptor signal regulatory protein alpha (SIRPalpha) counteracts macrophage phagocytosis of CD47-expressing host cells. As platelets also express CD47, we asked whether inhibitory CD47/SIRPalpha signaling regulates normal platelet turnover and clearance of platelets in immune thrombocytopenic purpura (ITP). CD47(-/-) mice had a mild spontaneous thrombocytopenia, which was not due to a decreased platelet half-life as a result of increased expression of P-selectin, CD61, or phosphatidylserine. In contrast, CD47(-/-) platelets were rapidly cleared when transfused into CD47(+/+) recipients, whereas CD47(+/-) platelets had a nearly normal half-life in CD47(+/+) mice under nonautoimmune conditions. CD47(-/-) mice were more sensitive to ITP, as compared with CD47(+/+) mice. In vitro, macrophage phagocytosis of immunoglobulin G (IgG)-opsonized CD47(-/-) platelets was significantly higher than that for equally opsonized CD47(+/+) platelets. However, when SIRPalpha was blocked, phagocytosis of CD47(+/+) platelets increased to the level of CD47(-/-) platelets. Phagocytosis of opsonized CD47(+/-) platelets was higher than that for CD47(+/+) platelets, but lower than that for CD47(-/-) platelets, suggesting a gene-dose effect of CD47 in this system. In conclusion, we suggest that inhibitory CD47/SIRPalpha signaling is involved in regulating platelet phagocytosis in ITP, and that targeting SIRPalpha may be a new means of reducing platelet clearance in ITP.

Animals↗
PubMed1986

Platelet fibrinogen binding in diabetes mellitus. Differences between binding to platelets from nonretinopathic and retinopathic diabetic patients.

While it is known that platelets from diabetic patients bind more fibrinogen than do platelets from normal subjects, there has been no study comparing this phenomenon in platelets from nonretinopathic and retinopathic patients. We have made such a comparison and have found the following. In agreement with previous reports, platelets from nonretinopathic diabetic patients bind abnormally high amounts of fibrinogen. No differences in the amount of fibrinogen bound to platelets (stimulated by collagen or thrombin) were found when data from nonretinopathic and retinopathic patients were compared. However, while aspirin (an inhibitor of thromboxane synthesis) reduced the abnormally high fibrinogen binding of platelets from nonretinopathic patients to normal control levels, it did not normalize the high fibrinogen binding of platelets from retinopathic diabetic patients. The combination of aspirin plus apyrase (an ADP scavenger) almost suppressed fibrinogen binding and aggregation of platelets from normal or nonretinopathic diabetic subjects, whereas it had a somewhat lesser effect on binding and aggregation of platelets from retinopathic subjects. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was the same in platelets from normal as well as nonretinopathic diabetic subjects and that this antibody could suppress the binding of fibrinogen and the aggregation of platelets from both types of patients just as it did in platelets from normal subjects. Thus, our data indicate that, while platelets from both retinopathic and nonretinopathic patients are hyperaggregable and show abnormally high binding of fibrinogen, they differ in that these abnormalities can be normalized in platelets from nonretinopathic patients by suppressing prostaglandin/thromboxane formation and scavenging ADP, but not in those from retinopathic patients.

Adolescent↗
PubMed1992

The influence of platelet-graft interaction on platelet survival in patients with aortobifemoral Dacron grafts.

In patients with arterial grafts, platelet consumption may be due either to platelet interaction with the graft, and/or concomitant platelet consumption in the rest of the arterial tree. This hypothesis was tested by quantifying the kinetics and platelet-graft interaction of indium-111-labelled platelets with double velour Dacron grafts in 13 patients with arterial insufficiency ascribed to atherosclerosis. Mean platelet lifespan (MPLS), 149 +/- 46 hours, was significantly shorter (P = 0.001) than normal. Labelled platelets were transiently deposited onto the graft surfaces. Peak 111In deposition on the grafts, 1.33 +/- 1.02% of injected labelled platelets, was reached at 70 +/- 33 hours. Thereafter the graft-platelet radioactivity decreased in parallel with platelet radioactivity in the circulation. There was no statistical correlation between MPLS and the factors known to be associated with graft platelet deposition: graft size; peak graft radioactivity; and the time to attain peak graft radioactivity. It is therefore concluded that in patients with arterial disease requiring graft implantation, the observed increased platelet consumption cannot only be ascribed to the interaction of platelets with the graft. Concomitant atherosclerosis is probably the important modifier of platelet consumption. The significant contribution of this factor to the shortening of the MPLS should be taken into account when assessing, by measuring platelet lifespan, the thrombogenicity of grafts.

Aged↗
PubMed2006

[Attempts for aspirin monitoring with a new assay system, Ultegra Rapid Platelet Function Assay (RPFA), based on turbidimetric platelet agglutination of whole blood samples].

Platelet aggregation measured by the optical density method has been applied for the assessment of platelet functions. However, as the method has to use platelet-rich plasma, it requires centrifugation of blood samples, which takes a considerable period of time. Using whole blood as samples has advantage because there is no pre-treatment of samples before measurement of platelet functions. Additionally, it would be desirable to have a bedside assay that reflects hyper-function of platelets and can monitor inhibitory effects of anti-platelet drugs. Rapid Platelet Function Assay (RPFA) is a qualitative test to aid the detection of platelet dysfunction due to anti-platelet drug ingestion, which uses citrated whole blood for sample in point of care or laboratory settings. The RPFA is a turbidimetric analysis, based on an optical detection system which measures platelet agglutination as an increase in light transmission. The aim of this study is to assess the accuracy and reproducibility of RPFA and to determine whether RPFA can monitor the effects of anti-platelet drugs. During the first 30 minutes after blood collection, Aspirin Reaction Units (ARU) determined with RPFA gradually increased, and reached its plateau after 30 minutes. The ARU values remained almost constant thereafter until 6 hours after blood collection (Fig. 3). These findings suggest that platelet function is unstable immediately after blood collection. Therefore, in this study ARU measurement was performed 60 minutes after blood collection. The reproducibility of ARU is very good both before and after aspirin intake. Seven days after daily uptake aspirin, ARU was decreased as compared with the control (440.8 +/- 39.4 vs. 663.4 +/- 2.4 ARU). RPFA measurement provides rapid information on platelet function that mirrors turbidimetric platelet agglutination and reflects COX1-depedent platelet activity.

Aspirin↗
PubMed1990

Platelet aggregation, platelet cAMP levels and thromboxane B2 synthesis in patients with diabetes mellitus.

Platelet aggregation, platelet cAMP levels and thromboxane B2 (TXB2) synthesis had been investigated in 40 diabetics (20 with microangiopathy and 20 without) and 24 normal controls. The washed platelets, but not platelet rich plasma (PRP), from the diabetics show greater sensitivity to aggregation in response to thrombin, collagen and arachidonic acid than controls (P less than 0.05). Platelets from the diabetics contain the significantly decreased cAMP levels (P less than 0.01) and synthesize the significantly greater amount of TXB2 (P less than 0.01) when induced by thrombin or collagen. Conversion of exogenously added arachidonic acid to TXB2 remained unchanged (P greater than 0.05). cAMP levels in platelets from the diabetics exhibited a significant negative linear correlation with thrombin- and collagen-induced TXB2 synthesis. There was no significant difference in platelet aggregation, platelet cAMP levels and platelet TXB2 synthesis between the diabetics with and without microangiopathy. It was suggested that in the diabetic platelets: The observed increase in platelet thromboxane A2 (TXA2) synthesis should be due to the increased activity of arachidonic acid-metabolizing system, most likely at phospholipase site; the elevated platelet TXA2 levels should inhibit platelet membrane-associated adenylate cyclase which lowered the cAMP levels in platelets; and this alternation should be the mechanism of platelet hyperaggregability, which might contribute in some way to diabetic microangiopathy.

Adult↗
PubMed1986

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor.

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

Antibodies, Monoclonal↗
PubMed1995

The effect of streptozocin-induced diabetes on platelet aggregation as determined by impedance aggregometry and platelet secretion: a possible role for nitric oxide.

Previous studies from our laboratory showed that diabetes increases platelet aggregation in rats, but only in washed platelets. In the present study, we evaluated platelet aggregation coupled with adenosine triphosphate (ATP) release in normal and diabetic rats using a whole blood electrical aggregometer. Additionally, we investigated the role of endothelium-derived relaxing factor, or nitric oxide, in the platelet reactivity in diabetic rats. Rats were made diabetic using streptozocin, 55 mg/kg. After 1 month the rats were anesthetized, and arterial blood samples were collected directly into sodium citrate solution 3.8%. Platelet counts and mean platelet volumes were determined. Platelet aggregation, disaggregation, and ATP release in response to adenosine diphosphate (ADP), collagen, and arachidonic acid were measured. Platelet aggregation in response to ADP, collagen, and arachidonic acid was not different in diabetic rats when compared with that in controls. However, ATP release in response to ADP was significantly increased in diabetic rats. Platelets from diabetic animals were significantly larger than those of controls. Nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, decreased platelet aggregation in response to ADP in the normal animals, but not in the diabetic animals. However, arginine-induced nitric oxide production decreased platelet aggregation and enhanced the occurrence of disaggregation in platelets from both normal and diabetic rats. From these studies, we conclude that platelet aggregation in diabetic rats is not different using whole blood electrical aggregation, but platelet ATP secretion is significantly enhanced. Additionally, nitric oxide may modulate the platelet aggregation, disaggregation, and ATP secretory response. However, it does not appear to be a major factor in the altered aggregation responses in diabetic rats.

Adenosine Diphosphate↗
PubMed1998

Platelet-derived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.

We investigated the role of the thrombin-activated platelet in modulating the rate and extent of activated protein C (APC)-catalyzed inactivation of platelet-derived factor Va and factor VaLeiden. Platelet-derived factor Va and factor VaLeiden were inactivated by APC at near identical rates; however, complete inactivation of the cofactors was never achieved. Greater residual cofactor activity remained when using thrombin-activated platelets compared with that observed with synthetic phospholipid vesicles and platelet-derived microparticles, suggesting that thrombin-activated platelets protect the cofactors from APC-catalyzed inactivation. This apparent protection was not due to (1) an insufficient number of membrane binding sites for APC or factor Va; (2) the destruction of these sites; or (3) the presence of a platelet-associated APC inhibitor. Results from a plasma-based clotting assay (with or without APC) with platelets or PCPS vesicles added to induce clot formation indicated that, even in the presence of high concentrations of APC, platelets offered protection of the cofactor by delaying cleavage at Arg506. This resulted in incomplete proteolysis of the heavy chain, suggesting that platelets can also protect plasma-derived factor Va from APC-catalyzed inactivation. However, additional experiments indicated that the plasma-derived cofactor, bound to thrombin-activated platelets, was completely inactivated by APC, suggesting that the plasma and platelet-derived cofactor pools represent different substrates for APC. Collectively, these results indicate that platelets sustain procoagulant events by providing a membrane surface that delays cofactor inactivation and by releasing a cofactor molecule that displays an APC resistant phenotype. Thus, at sites of arterial injury, the factor VLeiden mutation may not as readily predict arterial thrombosis, because the normal and variant platelet-derived cofactors are equally resistant to APC at the activated platelet surface.

Blood Platelets↗
PubMed1984

Platelet factor 4 does not promote von Willebrand factor binding to human platelets.

Ristocetin, protamine and Polybrene promote factor VIII:vWF binding and agglutination of formalinized platelets. It has been suggested that these polycations neutralize platelet negative surface charges and promote the attachment of VIII:vWF to platelets. Platelet factor 4 (PF4), protamine, and Polybrene inhibit heparin activity by neutralizing heparin negative charges. We tested the hypothesis that PF4, which is bound to the platelet surface after platelet activation and secretion, could promote the binding of VIII:vWF and subsequent platelet agglutination. Purified PF4 in concentrations comparable to those of ristocetin did not agglutinate formalinized platelets or induce the disappearance of VIII:vWF from the suspending plasma. Platelets were thrombin-treated in order to induce the release of PF4, and then formalinized and resuspended in normal plasma. These platelets did not agglutinate spontaneously, or at lower ristocetin concentrations than platelets that were not treated with thrombin before formalin fixation. Platelets were also activated by thrombin in the presence of EDTA to prevent surface binding of VIII:vWF or secreted PF4, and then formalinized. These platelets did not bind VIII:vWF in the presence of purified PF4. We conclude that even though PF4 binds to both polyanions and the platelet membrane, it does not promote the attachment of VIII:vWF.

Blood Coagulation Factors↗
PubMed2000

Human anti-heparin-platelet factor 4 antibodies are capable of activating primate platelets: towards the development of a HIT model in primates.

In the first step to establish an animal model of heparin-induced thrombocytopenia (HIT) that is physiologically relevant to humans, studies were undertaken to determine the similarities or differences between human and non-human primate (Macaca mulatta) platelets in HIT assay systems. The collagen-, ADP-, and TRAP-induced platelet aggregation, and flow cytometric analysis of P-selectin expression and microparticle formation were similar for both species platelets (p>0.1, n=18 each). The classical HIT assays using platelet-rich plasma (PRP) as well as a flow cytometric assay revealed the activation/aggregation and serotonin release assay (SRA) profiles for both primate and human platelets were similar in response to human HIT positive sera. All assays were heparin concentration-dependent; heparin, at 0.1 U/mL, produced maximum and similar platelet activation/aggregation and SRA responses with both primate (76+/-7%, n=18) and human (68+/-11%, n=20; p>0.1) platelets. At concentrations > or =10 U/mL, heparin suppressed the platelet aggregation and SRA responses in both systems. Primate and human platelets displayed similar behavior to low molecular weight heparin and pentasaccahride in HIT assay systems. Immunoglobulins isolated from serum of patients with HIT caused activation/aggregation of human (65+/-18%, n=10 donors) and primate (79+/-12%, n=6 monkeys, p>0.08) platelets. Unlike human platelets, the primate platelets exhibited a more consistent aggregation/release response (15 out of 18 primate platelets reactive). In contrast, human donors showed wide variations in the activation/release response (4 out of 10 reactive). These observations suggest that primate platelets are activatable by anti-H-PF4 antibodies, and support the hypothesis that primates can be used to develop an animal model to study the pathogenesis of HIT.

Animals↗
PubMed2002

Platelet GPIIb/IIIa is activated and platelet-leukocyte coaggregates formed in vivo during hemodialysis.

BACKGROUND/AIM: During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material. METHOD: We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P. RESULTS: Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes. CONCLUSION: This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.

Adult↗
PubMed1995

Platelet aggregation at high shear is impaired in patients with congenital defects of platelet secretion and is corrected by DDAVP: correlation with the bleeding time.

Techniques measuring platelet aggregation in vitro under the high shear rate conditions that can be found in the microcirculation could reflect the status of primary hemostasis better than the turbidimetric technique. We studied platelet aggregation at high shear in patients with prolonged bleeding time caused by congenital platelet secretion defects such as delta-storage pool deficiency and primary secretion defect. Two different techniques were used: shear-induced platelet aggregation in a cone-and-plate viscometer and the filter aggregation test. With both techniques, platelet aggregation at high shear rate was defective in 14 patients with delta-storage pool deficiency and in 8 with primary secretion defect. There was a statistically significant correlation between platelet aggregation at high shear rate and the bleeding time. In patients with delta-storage pool deficiency, platelet aggregation at high shear rate and the bleeding time were significantly correlated with the platelet serotonin content. The intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) (0.3 micrograms/kg) increased the plasma concentration of von Willebrand factor (vWf), shortened the bleeding time, and potentiated platelet aggregation at high shear rate in all patients. Because platelet aggregation at high shear rate requires vWf, the effect of DDAVP is probably due to the induced increase in plasma vWf. Therefore, platelet aggregation at high shear rate is defective in patients with congenital defects of platelet secretion and is potentiated by DDAVP. Potentiation of platelet aggregation at high shear rate may be one mechanism by which DDAVP shortens the prolonged bleeding time of patients with congenital defects of platelet secretion.

Bleeding Time↗