Formation Of Large Platelet Aggregates Research Articles

Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats.

Sepsis is often accompanied with thrombocytopenia partly due to platelet sequestration in the lung and liver. The spleen can store up to one-third of circulating platelets and can also significantly affect platelet transfusion outcomes by accumulating platelets. However, in sepsis, it is not clear whether there are platelet changes in the spleen which could contribute to sepsis-associated thrombocytopenia and also influence platelet transfusion outcomes. By using confocal microscopy, we examined endogenous rat platelets and infused human platelets in the spleen of severe combined immune deficient Rag2 KO rats which were injected intraperitoneally with lipopolysaccharide (LPS). LPS-injected Rag2 KO rats developed sepsis as indicated by increased TNFa, IL-6, IL-1b, and IL-10 levels and thrombocytopenia. Large platelet aggregates were observed in the spleen with majority located in the marginal zone and closely associated with CD169+ macrophages. Depletion of macrophages by clodrosome resulted in reduction of LPS-induced cytokine generation and alleviated LPS-induced thrombocytopenia. Macrophage depletion also remarkedly diminished large platelet aggregate formation in the splenic marginal zone but had less effect on those in red pulp. Infusion of human platelets into LPS-injected rats failed to raise platelet counts in the peripheral blood. In LPS-injected rat spleen, human platelets interacted with aggregated rat platelets in the marginal zone. In contrast, human platelets infused into control rats were located outside of splenic marginal zone. This study provides morphological evidence of platelet aggregates in the splenic marginal zone in sepsis which can interact with infused platelets and thus can contribute to platelet infusion refractoriness in sepsis. It indicates that macrophages play an important role in LPS-associated thrombocytopenia. It also suggests that CD169+ macrophages support platelet aggregate formation in the splenic marginal zone.

The Critical Role of Kindlin-3 in Intiation of Physiological Thrombus Formation ~ Analysis of Kindlin-3 Deficient Patient

Background: Kindlin-3 which is expressed mainly in hematopoietic cells, is essential for platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) activation and kindlin-3 deficiency causes severe bleeding problems. αIIbβ3 activation is tightly regulated by inside-out signaling, and the direct interaction of talin and kindlin-3 with β3-cytoplasmic tail following CalDAG-GEFI-induced Rap1 activation is critical for αIIbβ3 activation. We have reported that immediate and sustained αIIbβ3 activation by inside-out signaling is important for thrombus formation under physiological conditions (Blood 2016). However, the details of inside-out signaling are not still fully understood. Recently we identified patient with kindlin-3 deficiency as the first Japanese patient with kindlin-3 deficiency caused by novel p.W277X nonsense mutation. To clarify the role of kindlin-3 and the molecular mechanism of inside-out signaling, we analyzed single platelet behavior adhered on collagen under physiological flow conditions, and established kindlin-3 deficient human erythroleukemia HEL cell line. Case: The patient was 8-month old female, born to Japanese consanguineous parents. She has been suffering from bleeding tendency shortly after birth. Her peripheral blood showed slightly decreased platelet count (95x103/L), anemia (Hb 6.9 g/L), and elevated leukocyte count of 37.2x106/L with 1.0% blast. Although the surface expressions of glycoproteins were comparable to healthy control, her platelet aggregations and αIIbβ3 activation were strongly impaired in all agonist stimulations due to defective kindlin-3 expression. The sequencing analysis revealed the homozygous novel nonsense mutation, p.W277X (c.918G>A) in kindlin-3. Methods: Human platelets were obtained from healthy control subjects (CT) and patients with kindlin-3 deficiency and Glanzmann thrombasthenia (GT). Whole blood was perfused at a shear rate 1,250s-1 on collagen surface and shear-induced in vitro thrombus formation during 10 minutes was observed. To evaluate the single platelet behavior after the initial attachment on collagen, each single platelet was analyzed by CellTracker software (Pccinini F. et al. Bioinformatics 2015). To further determine the role of kindlin-3 in inside-out signaling, Kindlin-3 was knocked out by CRISPR-Cas9 system and established kindlin-3 deficient HEL cell line. Results: First, we compared shear-induced thrombus formation between CT, GT, and kindlin-3 deficiency. In contrast to the stable and large platelet aggregate formation in CT after 10 minutes blood perfusion, almost no aggregate was observed in both GT and Kindlin-3 deficiency. In kindlin-3 deficiency, the initial platelet attachment on collagen seemed comparable to that of CT and GT. However, the single adherent platelets looked unstable. Next, we determined the behavior of initially attached platelets. Between CT, GT, and kindlin-3 deficiency, the numbers of platelets attached on collagen during 10 seconds were comparable. Between the initially attached platelets, 31.25% of CT platelets formed stable adhesion followed by platelet aggregation. Similar to CT, 33% of GT platelets adhered stably. However, these GT platelets did not proceed to aggregate formation due to αIIbβ3 deficiency. In contrast to GT, kindlin-3 deficient platelets showed increased detachment, only 9% of initially attached platelets stably adhered. These results suggest that kindlin-3 is indispensable for platelet initial adhesion to collagen by integrin α2β1 activation and explains severe bleeding symptoms in kindlin-3 deficiency than GT. To further investigate how kindlin-3 contributes in initial platelet adhesion to collagen, we established kindlin-3 deficient HEL cell line. In contrast to parental HEL cells, PMA stimulation did not induce αIIbβ3 activation in kindlin-3 deficient HEL cells suggesting impaired inside-out signaling. The introduction of wild type kindlin-3 cDNA, but not W227X mutant, rescued αIIbβ3 activation. Conclusion: The detailed analyses by tracking single adherent platelet on collagen confirmed the role of kindlin-3 in initial step of physiological thrombus formation. Newly established kindlin-3 deficient HEL cell line is useful for further exploration of the role of kindlin-3 and inside-out signaling in platelets and expected contribution for development of new antiplatelet therapy. Disclosures Tomiyama: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Kyowa-Kirin: Honoraria.

Rho-kinase regulates human platelet activation induced by thromboxane A2 independently of p38 MAP kinase

We have previously demonstrated that ristocetin, an activator of GPIb/IX/V, induces the release of soluble CD40 ligand (sCD40L) via thromboxane A2 production in human platelets. It has been shown that thromboxane A2 induces the activation of Rho-kinase, a downstream effector of Rho, in human platelets. In the present study, we investigated the exact roles of Rho-kinase in thromboxane A2-induced platelet activation. We found that U46619, a thromboxane receptor (TP) agonist, induced the phosphorylation of cofilin, a target of Rho-kinase signaling, and that the cofilin phosphorylation by U46619 was suppressed by Y27632 or fasudil, specific inhibitors of Rho-kinase. Y27632 and fasudil markedly decreased large platelet aggregate formation by U46619. The release of sCD40L and secretion of platelet-derived growth factor (PDGF)-AB stimulated by U46619 were inhibited by Y27632 and fasudil. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, reduced the sCD40L release and PDGF-AB secretion. Y27632 and fasudil failed to affect the phosphorylation of p38 MAP kinase whereas SB203580 had little effect on the phosphorylation of cofilin induced by U46619. In conclusion, our results strongly suggest that Rho-kinase regulates thromboxane A2-induced human platelet activation independently of p38 MAP kinase.

Histones induce rapid and profound thrombocytopenia in mice

AbstractHistones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbβ3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbβ3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage.

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm 2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Human Antibodies with Specificity for the Spacer Domain of ADAMTS13 Are Derived from VH1-69 Germline Genes.

Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by the presence of circulating antibodies directed towards ADAMTS13. This results in the formation of large platelet aggregates that occlude the microvasculature giving rise to thrombocytopenia and micro-angiopathic hemolytic anemia in patients with TTP who lack functional ADAMTS13. Antibodies directed towards the spacer domain of ADAMTS13 are present in the majority of patients with acquired TTP. We have previously isolated a panel of human monoclonal antibodies directed towards ADAMTS13 from the immunoglobulin repertoire of one patient with acquired TTP employing phage display. To further extend these observations we obtained a panel of human anti-ADAMTS13 antibodies from a second patient with acquired TTP. Sequence analysis revealed that the variable heavy chain domains of the majority of anti-ADAMTS13 antibodies were encoded by germline gene segment VH1–69. The VH1–69 encoded antibody II-1 and the previously described antibody I-9, which was also encoded by VH1–69 were selected for further study. Epitope-mapping studies employing a hybrid ADAMTS13 variant containing the spacer domain of ADAMTS1 showed that both antibodies were directed towards the spacer domain. Both antibodies inhibited ADAMTS13 activity as measured by cleavage of FRETS-VWF73 substrate and also inhibited cleavage of VWF multimers under static conditions as well as under flow on the surface of endothelial cells. We subsequently developed an assay to monitor the presence of VH1-69 encoded antibodies in plasma of patients with acquired TTP using the anti-idiotypic monoclonal antibody G8 which specifically reacts with human antibodies that use the VH1-69 gene segment. Analysis of a large cohort of patients with acquired TTP revealed that antibodies reactive with the spacer domain of ADAMTS13 express the VH1-69 derived idiotype that is recognized by monoclonal antibody G8. These results support the notion that VH1-69 derived human antibodies directed towards the spacer domain of ADAMTS13 develop in the majority of patients with acquired TTP.

Serotoninergic Mechanisms Play a Major Role on Platelet-Mediated Thrombogenicity: Studies under Flow Conditions and Effects of Inhibitory Strategies.

Serotoninergic mechanisms are reciprocally implicated in depression and cardiovascular disorders. Serotonin (5-HT) may facilitate the development of a subpopulation of platelets with increased procoagulant activity. We have investigated the involvement of serotoninergic mechanisms in adhesive, cohesive and procoagulant function of platelets. Furthermore, we evaluated the antithrombotic properties of selective serotonin reuptake inhibitors (SSRI). For these purposes we used a series of experimental strategies including standard aggregometry, flow cytometry, perfusion techniques and determination of coagulation parameters. Serotonin concentrations ranging 0.5–5 μM were used in these studies. Citalopram, a selective serotonin reuptake inhibitor (SSRI), was used at concentrations equivalent to those reached in clinical practice.Aggregation studies indicate that 5-HT is a weak agonist for platelets in comparison with standard activating agents. Only the highest concentrations of 5-HT tested (5 μM) caused minimal and reversible platelet aggregation. Despite this modest effect on aggregation, 5-HT potentiated the aggregation induced by low concentrations of ADP (0.5 μM) in PRP samples (16.3±5.9 % vs. 28.1±5.5 %). Potentiation of aggegation was more evident when PRP was obtained from blood anticoagulated with LMWH (p<0.05), where some thrombin could be generated. Citalopram at 0.6 μM caused statistical reductions in % of maximal aggregation induced by ADP (2 or 0.5 μM) and COL (2.5 μg/ml) (p<0.05). Platelet aggregation was maximally inhibited with citalopram at concentrations exceeding clinical doses (6 μM). Flow cytometry studies revealed a mild increase of P-selectin expression in washed platelets in the first minute after activation with 5-HT. In isolated platelets, 5-HT produced irreversible aggregation when studies were performed in the presence of human-TF enriched microvesicles. A significant increase in Annexin V binding and presence of factor Va was detected by flow cytometry in the latter studies. Further studies performed in perfusion models using human blood flowing though chambers exposing a surface rich in collagen and TF demonstrated that presence of 5-HT in the perfusates increased thrombin generation (p<0.05) as demonstrated by elevations of F1+2 levels. Presence of citalopram in the perfusates significantly inhibited the formation of large platelet aggregates on the perfused thrombogenic surfaces (20.2±3.4 % vs. 11.5±1.7 %; p<0.05) and prevented elevations of F1+2 levels observed in previous studies. Our studies confirm that 5-HT is a weak agonist for platelets, though it possesses a marked ability to enhance the activation induced by other agonists. Moreover, 5-HT showed a powerful capacity to potentiate procoagulant responses of platelets under different experimental conditions and to enhance the thrombogenesis on damaged vascular surfaces. The fact that SSRI inhibited the responses elicited by 5-HT in the previous situations further confirm the implications of serotoninergic mechanisms in platelet activation. Our data reinforce the opinion that serotoninergic mechanisms play an important role in platelet-mediated thrombogenicity, suggesting that modulation of 5-HT mediated responses may offer a new potential target for the development of more powerful antithrombotic drugs.

Effect of ADP and ATP Receptor Antagonism on Erythrocytosis-Induced Platelet Aggregate Formation under Dynamic Flow Conditions.

Red blood cells (RBC) contribute significantly to hemostasis and thrombosis under oscillatory flow conditions, and erythrocytosis has been associated with increased thrombotic risk. To capture dynamic effects of RBC on platelets, we used a recently described Cone and Plate (let) Analyzer (CPA), to evaluate the effect of hematocrit (Hct) on platelet function in whole blood under arterial flow conditions (1800 sec-1, 2 min, 25°C). Anticoagulated blood, reconstituted to varying hematocrits with autologous RBC, demonstrated a significant increase (30–50%, n= 12, p < 0.05) in adherent platelet aggregate size at Hct levels >45%. No significant effect on platelet adhesion was noted. Increases in aggregate size were not accompanied by significant platelet dense or alpha granule secretion, measured using 14C-serotonin release and Platelet Factor 4 ELISA assays, respectively. However, when cell-free supernatants (SNT) were prepared post shearing to investigate the presence of proaggregatory activity by adding SNT to fresh PRP (1:10 SNT: PRP ratio), and quantifying the formation of small platelet aggregates with a laser light scatter aggregometer, SNT derived from sheared high Hct samples contained nearly twice (1.75 + 0.16, n=5, p < 0.05) the activity of sheared normal Hct samples. The erythrocytosis-induced increase in platelet aggregate size was not affected by pretreatment of blood with 0.05 mM aspirin, but could be prevented completely by antagonism of P2Y1 (MRS2179, 200 μM), P2Y12 (2-methylthioadenosine 5′-monophosphate, 10–100 μM), or P2X1 (MRS2159, 200 μM), ADP and ATP receptors, respectively, as well as by converting exogenous ADP to ATP with a combination of creatine phosphate (2 mM) and creatine phosphokinase (20 U/ml). Whereas negligible platelet granule secretion was measured post shear, metabolic inhibition of RBC with 1% sodium azide or 0.25% glutaraldehyde fixation (2 h, 37°C) fully inhibited erythrocytosis-enhanced increases in platelet aggregate size. Thus, the adenine nucleotides contributing to erythrocytosis-enhanced platelet aggregate formation under physiologic shear appear to be derived predominantly from RBC. Taken together, the data suggest that ADP and ATP are both required for erythrocytosis-induced large platelet aggregate formation, but that the presence of ATP alone is not sufficient. The results may further suggest a role for direct ADP or ATP receptor blockade for the prevention of erythrocytosis-associated thrombotic complications.

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen

Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb-IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb-IIIa and P-selectin than those on VWF. Activation of GpIIb-IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb-IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.

The Subendothelium of the HMEC-1 Cell Line Supports Thrombus Formation in the Absence of von Willebrand Factor and Collagen Types I, III and VI

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1's ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1's ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2,600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC's ECM. Interestingly, large thrombi were formed at 2,600/s when HMEC-1's ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1's ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.

Factors that affect the size of platelet aggregates in epinephrine-induced activation: A study using the particle counting method based upon light scattering

Platelet aggregate size was determined with a newly-developed platelet aggregometer, PA-100 (KOWA), which can quantitatively evaluate the size and number of platelet aggregates by means of the particle counting method based upon light scattering. Epinephrine-induced platelet aggregation consists of two phases, the former characterized by the formation of small-sized aggregates (less than 100 cells), which is followed by the phase of large aggregate formation with concomitant decrease in the number of small aggregates. These findings suggest that small aggregates fuse to form large aggregates. Effects of various inhibitors and antibodies directed against platelet membrane glycoproteins were evaluated on the size of platelet aggregates induced by epinephrine. Cyclooxygenase inhibitors, thromboxane A 2 receptor antagonists, and a Na + H + exchanger inhibitor (ethylisopropylamiloride) inhibited the formation of large aggregates (more than 100 cells) but not that of small aggregates. Cytochalasin B, which interferes with microfilaments, suppressed large aggregate formation, whereas taxol, which reacts with microtubules, had no effects. Anti- GPIIb IIIa monoclonal antibody (MoAb) inhibited both the formation of small and large platelet aggregates, while antibodies directed against GPIb, thrombospondin, P-selectin, or PECAM-1 had no effects on platelet aggregate formation. These findings, taken together, suggest that intracellular alkalinization, thromboxane A 2 formation and microfilament rearrangement are prerequisites for large platelet aggregate formation. GPIIb IIIa is involved in the formation of small as well as large aggregates, but a membrane glycoprotein(s) responsible for the transition of small aggregates into large aggregates awaits to be determined.

Flow cytometric studies of platelet responses to shear stress in whole blood.

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates ("large" refers to particles larger than 10 microns in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Inhibition of Platelet Aggregation by an SLE-Derived Human Hybridoma Autoantibody Against an Activation-Dependent Antigen

Anti-platelet autoantibodies may be responsible for hematological complications in patients with systemic lupus erythematosus (SLE), but the mechanisms by which these antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma autoantibody, 9604, recognizes a surface antigen expressed on platelets activated by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited by EDTA, indicating partial dependence on divalent cations. It did not appear to be dependent on platelet secretion, nor did it directly affect alpha-granule or dense granule secretion. The protein antigen responsible for the binding of 9604 to activated platelets was characterized by Western blot and immunoprecipitation and shown to have a native molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32). Antibody 9604 had little or no effect on the shape change and the initial rate of primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation of stirred platelet suspensions (IC50 < or = 1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation (secondary aggregation), with a major shift to smaller microaggregates and singlets, was confirmed by direct particle count and sizing studies. The functional inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests one potential mechanism that may play a role in the hemostatic disorders found in SLE.

Mechanisms involved in platelet procoagulant response.

Blood platelets are essential to the normal hemostatic process. Vessel wall injury produces several platelet agonists which through specific membrane receptors elicit a variety of cellular responses. Shape change, activation of binding sites for fibrinogen and other adhesive molecules, and secretion of intracellular granule contents ensure the rapid formation of large platelet aggregates, which prevent further loss of blood from the injured vessel. In addition, an important platelet response is the surface exposure of specific phospholipids, providing a catalytic surface for the assembly of enzyme complexes of the coagulation cascade. This platelet procoagulant response leads to a dramatic increase in the rate of thrombin formation, which allows rapid formation of an insoluble meshwork of fibrin, required to consolidate the primary haemostatic plug. On the other hand, the same catalytic surface is also instrumental in negative feedback control of the coagulation cascade by activated protein C. Platelets are the primary source of procoagulant lipid surfaces, but other cells such as erythrocytes or endothelial cells may, sometimes, under pathological conditions- also become procoagulant.

Free Platelet Count and Size Distribution During C1q Inhibition of Collagen-Induced Platelet Aggregation

Electronic free platelet counting was more sensitive than turbidimetry to detect collagen-induced platelet activation in human platelet-rich plasma. Purified human C1q exhibited a greater inhibitory effect on collagen-induced platelet aggregation in turbidimetry than free platelet counting. Because the change from small to large platelet aggregates is responsible for the continuing increase in light transmission, C1q was likely more capable of blocking the formation of large platelet aggregates than the formation of small aggregates from single platelets. The rate of change by collagen in light transmission and free platelet count was reduced in the presence of C1q but the timing of the peak response remained the same. Electronic platelet sizing revealed that the volume of single platelets transiently increased during the turbidimetric "lag phase". The mean, mode and median volume of the remaining free platelets then decreased, suggesting a selective loss of large, functionally more active platelets and/or platelet degranulation. C1q had no effect on the volume increment during the "lag phase", but reduced the subsequent fall in the volume of free platelets.