Shear Stress-induced Platelet Aggregation Research Articles

Novel Antiplatelet Activity of Ginsenoside Re Through the Inhibition of High Shear Stress-Induced Platelet Aggregation.

Bleeding is one of the most serious side effects of antiplatelet drugs. Efforts have been made to find new antiplatelet agents without bleeding complications. Shear-induced platelet aggregation (SIPA) occurs only under pathological conditions and is a promising target for overcoming bleeding problems. This work demonstrates that the ginsenoside Re selectively inhibits platelet aggregation induced by high shear stress. Human platelets were exposed to high shear stress using microfluidic chip technology, and aggregation, activation, and phosphatidylserine (PS) exposure were measured. The Von Willebrand Ristocetin Cofactor (vWF:RCo) assay and western blot were used to evaluate the effect of the vWF-GPⅠb/PI3K/Akt signal pathway. The coagulation and bleeding risk were evaluated by measuring the coagulation parameters PT, APTT, TT, and thromboelastography. The 3-dimensional morphology of platelet aggregates was observed by a microscopic 3-dimensional imaging. Re was a potent inhibitor of SIPA, with an IC 50 of 0.071 mg/mL. It effectively blocked shear stress-induced platelet activation without any significant toxicity. It was highly selective against SIPA, effectively inhibiting vWF-GPIb and the downstream PI3K/Akt signaling pathway. Most importantly, Re did not affect normal blood coagulation and did not increase the risk of bleeding. In conclusion, Re inhibits platelet activation through the inhibition of the vWF-GPIb/PI3K/Akt pathway. Thus, it might be considered as a new antiplatelet drug in the prevention of thrombosis without increasing the risk of bleeding.

Water-soluble tomato concentrate modulates shear-induced platelet aggregation and blood flow in vitro and in vivo.

Water-soluble tomato concentrate (WSTC), extracted from mature tomatoes, is the first health product in Europe that has been approved “to help maintain normal platelet activity to maintain healthy blood flow.” We hypothesized that WSTC might exert an influence on blood flow shear stress-induced platelet aggregation (SIPA) and in turn maintains healthy blood flow. We used a microfluidic system to measure the effects of WSTC on SIPA in vitro. We also used the strenuous exercise rat model and the κ-carrageenan-induced rat tail thrombosis model to demonstrate the effects of WSTC on blood flow. WSTC significantly inhibited platelet aggregation at pathological high shear rate of 4,000 s–1 and 8,000 s–1 in vitro (P < 0.05 or P < 0.01). WSTC reduced the platelet adhesion rate and increased the rolling speed of platelets by inhibiting binding to Von Willebrand Factor (vWF) (P < 0.05 or P < 0.01). The oral administration of WSTC for 4 weeks in strenuous exercise rats alleviated hyper-reactivity of the platelets and led to a significant reduction in the plasma levels of catecholamine and IL-6. WSTC treatment also led to a reduction in black tail length, reduced blood flow pulse index (PI) and vascular resistance index (RI), and ameliorated local microcirculation perfusion in a rat model of thrombosis. WSTC exerted obvious inhibitory effects on the platelet aggregation induced by shear flow and alleviated the blood flow and microcirculation abnormities induced by an inflammatory reaction.

Regulation of Key Antiplatelet Pathways by Bioactive Compounds with Minimal Bleeding Risk.

Cardiovascular disease is strongly influenced by platelet activation. Platelet activation and thrombus formation at atherosclerotic plaque rupture sites is a dynamic process regulated by different signaling networks. Therefore, there are now focused efforts to search for novel bioactive compounds which target receptors and pathways in the platelet activation process while preserving normal hemostatic function. The antiplatelet activity of numerous fruits and vegetables and their multiple mechanisms of action have recently been highlighted. In this review, we review the antiplatelet actions of bioactive compounds via key pathways (protein disulfide isomerase, mitogen-activated protein kinases, mitochondrial function, cyclic adenosine monophosphate, Akt, and shear stress-induced platelet aggregation) with no effects on bleeding time. Therefore, targeting these pathways might lead to the development of effective antiplatelet strategies that do not increase the risk of bleeding.

Antithrombotic effect of SP-8008, a benzoic acid derivative, through the selective inhibition of shear stress-induced platelet aggregation.

Background and PurposeBleeding is one of the most critical adverse effects of antithrombotic drugs, and many efforts have been made to discover novel antiplatelet agents without bleeding complications. Shear stress‐induced platelet aggregation (SIPA), where the interaction of von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib constitutes the initial step, is a promising target to overcome bleeding problems, as SIPA occurs only in pathological conditions. Here, we describe SP‐8008, a novel modulator of vWF–GP Ib interactions and evaluated its antiplatelet/antithrombotic effects.Experimental ApproachNewly synthesized compounds were screened for antiplatelet effects in vitro, using human platelets exposed to high shear stress. Aggregation, intracellular calcium level, granule secretion, and integrin activation were assessed. Molecular modelling using virtual docking and flow cytometry were used to evaluate effects on vWF–GP Ib interactions. Antithrombotic effects in vivo were determined in rats, using arterial thrombosis and shear stress‐specific thrombosis. Transection tail bleeding time was used to evaluate adverse effects.Key ResultsSP‐8008 was a potent inhibitor of SIPA, with IC50 of 1.44 ± 0.09 μM. SP‐8008 effectively and broadly blocked shear stress‐induced platelet activation events, without any significant toxicity. Importantly, SP‐8008 was highly selective against SIPA, effectively interfering with vWF–GP Ib engagement. Most importantly, SP‐8008 exerted significant antithrombotic effects in vivo in both shear stress‐specific and arterial thrombosis, without prolonging bleeding time.Conclusions and ImplicationsOur results demonstrated that SP‐8008 can be a novel selective antiplatelet agent with improved safety profile.

Antithrombotic Effects of Paeoniflorin from Paeonia suffruticosa by Selective Inhibition on Shear Stress-Induced Platelet Aggregation.

Antiplatelet agents are important in the pharmacotherapeutic regime for many cardiovascular diseases, including thrombotic disorders. However, bleeding, the most serious adverse effect associated with current antiplatelet therapy, has led to many efforts to discover novel anti-platelet drugs without bleeding issues. Of note, shear stress-induced platelet aggregation (SIPA) is a promising target to overcome bleeding since SIPA happens only in pathological conditions. Accordingly, this study was carried out to discover antiplatelet agents selectively targeting SIPA. By screening various herbal extracts, Paeonia suffruticosa and its major bioactive constituent, paeoniflorin, were identified to have significant inhibitory effects against shear-induced aggregation in human platelets. The effects of paeoniflorin on intraplatelet calcium levels, platelet degranulation, and integrin activation in high shear stress conditions were evaluated by a range of in vitro experiments using human platelets. The inhibitory effect of paeoniflorin was determined to be highly selective against SIPA, through modulating von Willebrand Factor (vWF)-platelet glycoprotein Ib (GP Ib) interaction. The effects of paeoniflorin on platelet functions under high shear stress were confirmed in the ex vivo SIPA models in rats, showing the good accordance with the anti-SIPA effects on human platelets. Treatment with paeoniflorin significantly prevented arterial thrombosis in vivo from the dose of 10 mg/kg without prolonging bleeding time or blood clotting time in rats. Collectively, our results demonstrated that paeoniflorin can be a novel anti-platelet agent selectively targeting SIPA with an improved safety profile.

Cyclocurcumin from Curcuma longa selectively inhibits shear stress-induced platelet aggregation

Abstract Curcuma longa (C. longa) has been the subject of intensive research for pleiotropic therapeutic potential. However, except for curcumin, the biological activities of other active ingredients of C. longa are not well known. Here, we demonstrated that C. longa extract significantly attenuated shear stress-induced platelet aggregation and cyclocurcumin exhibited the most potent activity (IC50 of 6.33 ± 3.29 μM). Cyclocurcumin potently inhibited shear stress-induced platelet activation, primarily through modulating the initial step, vWF – platelets GP Ib interaction. Moreover, cyclocurcumin effectively inhibited thrombus formation, without influencing platelet cell viability or blood clotting time. Collectively, we demonstrated that cyclocurcumin might be a promising therapeutic agent for the treatment or prevention of thrombotic diseases.

Mutant botrocetin‐2 inhibits von Willebrand factor‐induced platelet agglutination

Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.

ADAMTS13 unbound to larger von Willebrand factor multimers in cryosupernatant: implications for selection of plasma preparations for thrombotic thrombocytopenic purpura treatment

Thrombotic thrombocytopenic purpura (TTP) is characterized by deficient ADAMTS13 activity. Treatment involves plasma exchange (PE). Both fresh-frozen plasma (FFP) and cryosupernatant (CSP) are used, but it remains to be determined which is more effective. To analyze the interaction between von Willebrand factor (VWF) and ADAMTS13, we used large-pore isoelectric focusing (IEF) analysis followed by detection with anti-ADAMTS13 monoclonal antibody. FFP, CSP, cryoprecipitate (CP), and purified ADAMTS13 were analyzed for their effects on high shear stress-induced platelet aggregation (H-SIPA). IEF analysis of normal plasma revealed three groups of ADAMTS13 bands with pI of 4.9 to 5.6, 5.8 to 6.7, and 7.0 or 7.5. Two band groups (pI 4.9-5.6 and 5.8-6.7) were found in plasma of a patient with Type 3 von Willebrand disease, in which VWF is absent, whereas no bands were found in plasma of a patient with congenital ADAMTS13 deficiency. Mixing these plasmas generated the bands at pI 7.0 or 7.5, representing the VWF-ADAMTS13 complex; these bands were absent in CSP. FFP and purified ADAMTS13 down regulated H-SIPA in a dose-dependent manner. However, CP did not inhibit H-SIPA in the initial phase, and the degree of inhibition at the endpoint was almost indistinguishable from those of the other two plasma products. Both plasma products (FFP and CSP) are effective for PE in TTP patients. However, CSP may be more favorable, because it has lower levels of VWF and almost normal ADAMTS13 activity, but lower levels of ADAMTS13 in complex with larger VWF multimers.

Novel Antiplatelet Activity of Protocatechuic Acid through the Inhibition of High Shear Stress-Induced Platelet Aggregation

Bleeding is the most common and serious adverse effect of currently available antiplatelet drugs. Many efforts are being made to develop novel antithrombotic agents without bleeding risks. Shear stress-induced platelet aggregation (SIPA), which occurs under abnormally high shear stress, plays a crucial role in the development of arterial thrombotic diseases. Here, we demonstrate that protocatechuic acid (PCA), a bioactive phytochemical from Lonicera (honeysuckle) flowers, selectively and potently inhibits high shear (>10,000 s(-1))-induced platelet aggregation. In isolated human platelets, PCA decreased SIPA and attenuated accompanying platelet activation, including intracellular calcium mobilization, granule secretion, and adhesion receptor expression. The anti-SIPA effect of PCA was mediated through blockade of von Willebrand factor binding to activated glycoprotein Ib, a primary and initial event for the accomplishment of SIPA. Conspicuously, PCA did not inhibit platelet aggregation induced by other endogenous agonists like collagen, thrombin, or ADP that are important in both pathological thrombosis and normal hemostasis. Antithrombotic effects of PCA were confirmed in vivo in a rat arterial thrombosis model, where PCA significantly delayed the arterial occlusion induced by FeCl(3). Of particular note, PCA did not increase bleeding times in a rat tail transection model, whereas conventional antiplatelet drugs, aspirin, and clopidogrel substantially prolonged it. Collectively, these results suggest that PCA may be a novel antiplatelet agent that can prevent thrombosis without increasing bleeding risks.

Migraine: Possible Role of Shear‐Induced Platelet Aggregation With Serotonin Release

Migraine patients are at an increased risk for stroke, as well as other thromboembolic events. This warrants further study of the role of platelets in a proportion of migraine patients. To extend the "platelet hypothesis" using literature data and observations made in a rat model of shear stress-induced platelet aggregation. Such aggregation causes release of serotonin, leading to vasoconstriction during sufficiently strong aggregation and to long-lasting vasodilation when aggregation diminishes. This vasodilation also depends on nitric oxide and prostaglandin formation. A role for platelet aggregation in a number of migraineurs is indicated by reports of an increased platelet activity during attacks and favorable effects of antiplatelet medication. We hypothesize that in those patients, a migraine attack with or without aura may both be caused by a rise in platelet-released plasma serotonin, albeit at different concentration. At high concentrations, serotonin may cause vasoconstriction and, consequently, the neuronal signs of aura, whereas at low concentrations, it may already stimulate perivascular pain fibers and cause vasodilation via local formation of nitric oxide, prostaglandins, and neuropeptides. Platelet aggregation may be unilaterally evoked by elevated shear stress in a stenotic cervico-cranial artery, by reversible vasoconstriction or by other cardiovascular abnormality, eg, a symptomatic patent foramen ovale. This most likely occurs when a migraine trigger has further enhanced platelet aggregability; literature shows that many triggers either stimulate platelets directly or reduce endogenous platelet antagonists like prostacyclin. New strategies for migraine medication and risk reduction of stroke are suggested.

Novel anti-platelet activity of protocatechuic acid through inhibition of shear stress-induced platelet aggregation

Novel anti-platelet activity of protocatechuic acid through inhibition of shear stress-induced platelet aggregation

Reduction in Platelet Activation by Citrate Anticoagulation Does Not Prevent Intradialytic Hemodynamic Instability

Background: The etiology of intradialytic hemodynamic instability is multifactorial. Of the various factors involved, a rise in core temperature seems to be crucial. In this respect, the bioincompatibility of hemodialysis (HD) treatment might play an important role. The application of cool dialysate reduces the number of periods of intradialytic hypotension (IDH) considerably. In rats, roller pump perfusion caused hypotension by shear stress induced platelet aggregation and subsequent serotonin release. During clinical HD, citrate anticoagulation abolished platelet activation almost completely. Hence, citrate anticoagulation might reduce IDH, whereas the beneficial effect of cool dialysate might be partly explained by reduced platelet activation. Methods: In the present study, blood pressure, IDH episodes, platelet activation, platelet aggregation, and serotonin release were studied crossover in 10 patients during HD with dalteparin anticoagulation at normal and low dialysate temperatures and during HD with citrate. Results: Citrate strongly reduced platelet activation, but did not improve IDH. The blood pressure was best preserved during cool-temperature HD, despite manifest platelet activation. Platelet activation was not accompanied by a rise in the plasma serotonin concentration. Conclusions: Three major conclusions can be drawn: (1) it is unlikely that platelet activation and subsequent serotonin release underlie IDH in the clinical situation; (2) the protective effects of cool dialysate on IDH appear to be independent of HD-induced platelet activation, and (3) extrapolating results from rat experiments to the human situation requires uppermost prudence.

Cyclooxygenase-2 Inhibitors Enhance Shear Stress-Induced Platelet Aggregation

We aimed to investigate the effect of parecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on in vivo shear stress-induced platelet aggregation in a rat model of arterial bypass with focal narrowing. Long-term use of COX-2 inhibitors is associated with increased incidence of adverse cardiovascular events, especially in patients with a history of cardiovascular disease. These patients are at risk for thrombotic occlusion of arterial stenoses initiated by shear stress-induced platelet aggregation. To mimic the combination of a tight arterial stenosis and high shear stress in rats, an extracorporeal shunt from carotid to femoral artery was compressed by the rollers of a pump. Platelet aggregation was continuously measured by a photometric detector in the shunt. Pretreatment with parecoxib (20 mg/kg) almost doubled shear stress-induced platelet aggregation (188% vs. 100% in control subjects, p = 0.0003). This was accompanied by a fall in plasma 6-keto-prostaglandin F(1alpha) from 100 +/- 25 pg/ml to 36 +/- 11 pg/ml (p < 0.0001). Enhanced platelet aggregation was also observed with high-dose aspirin (150 mg/kg) (146%; p = 0.02) but not with low-dose aspirin (25 mg/kg), which reduced aggregation (68%; p = 0.01). The effect of parecoxib was neutralized by low-dose (1 mg/kg) clopidogrel (from 188% to 92%; p = 0.0001), but not by low-dose aspirin (from 188% to 177%; p = NS). In the presence of an arterial stenosis, COX-2 inhibitors enhance shear stress-induced platelet aggregation. This enhancement was prevented by low-dose clopidogrel but not by low-dose aspirin. Clopidogrel might therefore allow COX-2 inhibitors to be used without raising risk of thrombotic occlusion.

Shear stress-induced pH increase in plasma is mediated by a decrease in PCO2: The increase in pH enhances shear stress-induced P-selectin expression in platelets

To investigate shear stress-induced platelet activation, the cone-plate viscometer or the Couette rotational viscometer has been widely used. In a previous report, it was shown that shearing platelet-rich plasma using a Couette rotational viscometer could lead to an increase in pH by CO2 release. However, any clear mechanism has not been provided. In this study, we examined whether shearing cell free plasma only using a cone-plate viscometer can also induce pH increase and studied the underlying mechanism of shear-induced pH increase by directly measuring total CO2 (TCO2) and CO2 tension (PCO2). When human plasma was sheared using a cone-plate viscometer, the pH of the human plasma increased time- and shear rate-dependently. Although TCO2 of human plasma was not affected, PCO2 was decreased by shearing, indicating that the decreased PCO2 is associated with a pH increase of plasma. In addition, the pH of bicarbonate-containing suspension buffer was also shown to be increased by shearing; suggesting that the platelet studies using suspension buffers containing bicarbonate could be affected similarly. The effects of pH changes on shear stress-induced platelet activation were also investigated in the same in vitro systems. While shear stress-induced platelet aggregation was not affected by the pH changes, P-selectin expression was significantly increased in accordance with the pH increase. In conclusion, shear stress using a cone-plate viscometer induces pH increase in plasma or bicarbonate-containing suspension buffer through a PCO2 decrease and the pH changes alone can contribute to platelet activation by enhancing shear stress-induced P-selectin expression.

Use of anti-thrombotic agents during chemotherapy for epithelial ovarian cancer

The association between malignancy and venous thrombotic events is well established. However, arterial thrombosis among cancer patients is extremely rarely reported. There are several mechanisms of arterial thrombosis or embolism in malignancy. Important mechanisms in arterial thrombogenesis are shear stress-induced platelet aggregation and platelet-derived microparticles. Both of these are induced by major abdominal surgery. A major abdominopelvic surgery followed by adjuvant platinum-based combined chemotherapy is routinely performed for epithelial ovarian cancer which is the leading cause of death among all gynecologic malignancies. These patients have a greater risk of arterial thrombosis at the postoperative period. If the affected arteries are relatively larger, clinical findings will be evident due to limb ischemia or fatal organ infarctions. However, thrombosis of the small arteries disturbs the tissue circulation which is extremely important for the chemotherapeutic agents to reach the residual tumor cells. When the thrombosis of small arteries is prevented, these drugs will reach all of the residual macroscopic or microscopic tumoral tissues and so the prognosis of the patients may be improved. Therefore, we hypothesize that anti-platelet therapy with aspirin is needed to be initiated during the postoperative period of epithelial ovarian cancer patients and be continued as long as chemotherapy goes on. Such an approach might have a role in optimizing the oncological prognosis of these patients via increasing the effectiveness of cytotoxic therapy since some of the recurrences may be caused by some microscopic tumor foci which were not affected by cytotoxic drugs because of subclinical small arterial thromboses.

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 ({4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine–glycine–aspartic acid–serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A (( S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 μg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

Shear stress-induced aggregation of oxidized platelets

Although shear stress-induced platelet aggregation (SIPA) has been noted, the shear stress-induced aggregation of oxidized platelets (SIOPA) has not been investigated. To investigate SIOPA, small quantity of hemoglobin (Hb) solution was first added to plasma to induce oxidation stress of platelets and then the plasma was sheared by a cone/plate viscometer at shear stress of 66 dyn/cm 2. In addition, to investigate the change in expression of the membrane receptors of glycoprotein Ib (GpIb) for oxidized platelets, mean fluorescence intensity (MFI) for those platelets was detected by flow cytometric technique. The results showed that the level of the oxidation stress of platelets, as presented in malondialdehyde (MDA) values, was well correlated with the quantities of added Hb. Moreover, the aggregation of SIOPA corresponded with the levels of oxidation stress of platelets. Platelets pretreated with aspirin could only partially reduce the aggregation of SIOPA. Similarly, aspirin pretreatment could partially reduce the lowering of MFI value that indicated downexpression of GpIb receptors. Furthermore, the aggregations of SIOPA corresponded with the lowered percentage of those MFI values for studied cases. We thereby suggested that the aggregation of SIOPA is related to both the level of oxidation stress and the downexpression of GpIb receptors for oxidized platelets.

Shear stress-induced platelet aggregation in children with minimal change nephrotic syndrome

Analysis of the hemostasis system using biochemical techniques in children with minimal change nephrotic syndrome (MCNS) has previously been restricted to in vitro assays. The recent introduction of measurement of shear stress-induced platelet aggregation (SIPA) using platelet-rich plasma (PRP) has facilitated detailed investigation of the hemostatic system in vivo. In order to further analyze the etiology of the thrombotic tendency exhibited by patients with childhood MCNS, we investigated SIPA at both low shear stress (L-SIPA) and high shear stress (H-SIPA) in 14 children with MCNS using PRP collected weekly after commencing prednisolone therapy. Seven patients were newly diagnosed cases of MCNS (ND) whereas the remainder had suffered a disease relapse (DR). Prior to prednisolone therapy L-SIPA, which was thought to be affected by fibrinogen (Fbg) levels, was significantly increased in both patient groups compared to normal controls (17.4 +/- 4.1% vs. 3.6 +/- 0.7%, ND vs control, p < 0.01: 11.7 +/- 3% vs. 2 +/- 0.7%, DR vs control, p < 0.01) with values declining at weekly intervals thereafter. Plasma Fbg levels in simultaneously collected samples followed a similar course. In contrast, H-SIPA, which was thought to be affected by von Willebrand factor (VWF), was significantly enhanced in MCNS patients after 1 week of prednisolone therapy compared to the controls (45 +/- 5.1% vs. 26.3 +/- 3.5%, ND vs normal, p < 0.05: 36.9 +/- 3.3% vs. 25.5 +/- 1.6%, DR vs. normal, p < 0.05). Plasma ristocetin cofactor and VWF antigen levels in simultaneously collected samples followed a similar course, whereas thrombin-antithrombin complex (TAT) levels remained constant. These results indicate that SIPA is enhanced in the acute stage of childhood MCNS, especially L-SIPA prior to the initiation of prednisolone therapy and H-SIPA after 1 week of prednisolone therapy, and that these phenomena may be affected by plasma Fbg and VWF levels, respectively. The enhanced SIPA may play an important thrombogenic role in the acute phase of childhood MCNS.

Report on the workshop: Von Willebrand factor and Thrombotic Thrombocytopenic Purpura.

INTRODUCTION (Dr. P. Ganguly)A workshop on von Willebrand factor (vWf) andThrombotic Thrombocytopenic Purpura (TTP) was orga-nized by the Division of Blood Diseases and Resourcesof the National Heart, Lung, and Blood Institute(NHLBI) and was held on July 31, 2000 at the Lister HillAuditorium on the National Institutes of Health campus.TTP is a difficult disease with a high mortality and treat-ment options are limited to plasma transfusion or ex-change. The objective of the workshop was to review thecurrent understanding of the pathogenesis of TTP and tostimulate new research and clinical advances.The agenda of the workshop was broadly divided intothree parts. The first group of lectures addressed the fun-damental aspects of vWf molecular structure, vWf bio-synthesis, vWf-platelet interactions, classification ofmetalloproteases, and general principles of antibody for-mation.The second group of presentations discussed the dys-function of the vWf-cleaving metalloprotease in TTP pa-tients, the specificity of this observation and correlationwith disease status, assay of the vWf-cleaving metallo-protease, and development of an autoantibody in somepatients with acute idiopathic TTP. The third session fo-cused on the management of patients with TTP and thehemolytic-uremic syndrome (HUS), and the possible roleof vWf in these conditions. In the following text, theWorkshop speakers on each topic are identified.VON WILLEBRAND FACTOR: SYNTHESIS,STRUCTURE AND FUNCTION (Drs. E. Sadler,D. Wagner, and Z. Ruggeri)The vWf gene is located on chromosome 12. The pri-mary translation product includes a large 741 amino acidpropeptide sequence located at the N-terminal end of themolecule.In the endoplasmic reticulum, pro-vWf dimers areformed by disulfide bonding between “cystine knots” atthe C-terminal ends of pro-vWf molecules. The pro-vWfdimers then polymerize (multimerize) by the formationof additional disulfide bonds at the N-terminal ends ofthe pro-vWf dimers. The propeptide may function as adisulfide isomerase to promote this multimerization ofpro-vWf dimers in the acidic environment of the trans-Golgi network. After multimerization, the propeptidesare removed by intra-cellular proteolysis. The multimersof mature vWf subunits are then further modified byglycosylation and sulfation. The largest vWf multimersproduced by this complicated synthetic machinery maycontain more than 40 subunits, and exceed 20 millionDaltons in molecular mass. The completed vWf multi-mers, along with the previously removed propeptide mol-ecules, are either secreted constitutively or stored in theWeibel-Palade bodies of endothelial cells (and thea-granules of megakaryocytes and platelets). It is fromthe Weibel-Palade bodies that the most biologically ac-tive large and unusually large vWf multimers are re-leased when endothelial cells are stimulated.vWf mediates the adhesion of platelets to sites of vas-cular damage and, as the carrier protein for blood clottingfactor VIII, is required for normal factor VIII survival inthe circulation. The binding site for factor VIII on vWfmultimers is located near the N-terminal regions of ma-ture vWf subunits. Each vWf subunit consists of 2,050amino acids organized into separate domains with differ-ent structures and functions. The critical binding site forplatelet glycoprotein(GP) Iba, for example, is containedwithin domain A1. Other domains encode binding sitesfor collagen and a binding site for platelet GP IIb-IIIacomplexes (integrin aIIbb3). This latter binding site con-tains the amino acid sequence Arg-Gly-Asp (a sequencethat also enables fibrinogen and fibronectin to bind GPIIb-lIIa).

Inhibition by combined therapy with ticlopidine and aspirin of enhanced platelet aggregation during physical exercise in patients with coronary artery disease

Background Strenuous exercise can be a major trigger for coronary thrombosis and it enhances platelet aggregation. Methods We evaluated the effect of antiplatelet therapy on shear stress–induced platelet aggregation (SIPA), in addition to agonist-induced aggregation, before and immediately after ergometer exercise in patients with stable coronary artery diseases (CAD). Forty-eight patients with stable CAD were randomly distributed into 3 groups: no antiplatelet drug (patient control, n = 16), aspirin (ASA) monotherapy (n = 16), and combined therapy with ticlopidine (TIC) and ASA (n = 16). Results There were significant increases in not only adenosine phosphate (ADP)– and collagen-induced platelet aggregation but also in SIPA during exercise by the patient control group. ASA monotherapy did not attenuate the enhanced ADP-induced aggregation nor SIPA. Combined ASA + TIC therapy significantly inhibited SIPA as well as ADP-induced aggregation both before and after exercise. Significant increases in levels of plasma von Willebrand factor (vWF) occurred during exercise, and these antiplatelet therapies had no apparent effect on increased vWF levels during exercise. Exercise induced a significant increase in the plasma thrombin-antithrombin III complex level with no significant changes in the level of plasmin-plasmin inhibitor complex level in all 3 groups. Conclusions Combined therapy with ASA + TIC effectively inhibited increased platelet aggregability in response to acute exercise, with no effects on coagulant or fibrinolytic potentials in patients with CAD. The data suggest that TIC combined with ASA may be superior to ASA alone in preventing acute coronary events during exercise in patients with coronary atherosclerotic disease. (Am Heart J 2001;142:e1.)