Activation-independent platelet adhesion and aggregation under elevated shear stress

Platelet aggregation, which contributes to bleeding arrest and also to thrombovascular disorders, is thought to initiate after signaling-induced activation. We found that this paradigm does not apply under blood flow conditions comparable to those existing in stenotic coronary arteries. Platelets interacting with immobilized von Willebrand factor (VWF) aggregate independently of activation when soluble VWF is present and the shear rate exceeds 10 000 s(-1) (shear stress = 400 dyn/cm(2)). Above this threshold, active A1 domains become exposed in soluble VWF multimers and can bind to glycoprotein Ibalpha, promoting additional platelet recruitment. Aggregates thus formed are unstable until the shear rate approaches 20 000 s(-1) (shear stress = 800 dyn/cm.(2)). Above this threshold, adherent platelets at the interface of surface-immobilized and membrane-bound VWF are stretched into elongated structures and become the core of aggregates that can persist on the surface for minutes. When isolated dimeric A1 domain is present instead of native VWF multimers, activation-independent platelet aggregation occurs without requiring shear stress above a threshold level, but aggregates never become firmly attached to the surface and progressively disaggregate as shear rate exceeds 6000 s(-1). Platelet and VWF modulation by hydrodynamic force is a mechanism for activation-independent aggregation that may support thrombotic arterial occlusion.     

The kinetics of platelet aggregation induced by fluid-shearing stress

The work herein examines in vitro the kinetics of platelet aggregation in response to the fluid-shearing stress imparted by a rotational viscometer. Aggregation is expressed through changes in particle size histograms and total particle count. Platelet aggregation commenced above 2000 sec-1 shear rate and was stable above 5000 sec-1. Activation by shear required less than 10 sec at all shear rates. At shear rates above 5000 sec-1, the particle count fell by one-half within 10 sec. By this point aggregates of 1000 platelets or more had appeared. Diluting the platelet suspensions suppressed shear-induced aggregation but not aggregation to ADP, supporting the hypothesis that shear-induced platelet aggregation proceeds through chemical release.     

Quantifying the effects of shear stress and shear exposure duration regulation on flow induced platelet activation and aggregation

A heightened flow shear stress magnitude or increased exposure duration at any shear stress magnitude enhances platelet activation. It has been suggested that increased circulating activated platelets are an indication of enhanced cardiovascular risk and that activated platelets can perpetuate cardiovascular diseases. However, the combined effects that shear stress and shear exposure duration have on platelet activation and aggregation are not clear. Our objective was to identify a new parameter (“shear stress-exposure time”) to predict platelet functional changes better than the absolute magnitude of shear stress. Platelets were exposed to shear stress waveforms, with different combinations of shear stress magnitude and exposure duration, in a circulation flow loop. Timed samples were removed from the flow loop to quantify thrombogenicity (modified prothrombinase assay), aggregation potential (optical platelet aggregometry), and cell surface marker expression (flow cytometry). Flow induced platelet activation and aggregation was enhanced with increased shear stress-exposure time. Platelets that were exposed to waveforms with the same shear stress-exposure time had the same thrombogenicity and aggregation potential. This was true even for waveforms that exposed platelets to shear stress magnitudes varying from 4 to 40 dynes/cm². These results indicate that shear stress-exposure time is a good predictor of platelet activation and aggregation levels and it may be able to predict the likelihood of cardiovascular disease onset.     

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.     

Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157.

The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays. Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress. Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml. This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter. In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP. 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3). Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not. Several cytokines produced from monocytes reproduced this event in vitro. Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1. This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels. Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues.     

Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments

Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWE All these fragments completely inhibited botrocetin-induced VWF binding to GPIb alpha at a final concentration of 40-200 microM. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 microM in the following order: 315 kD Fr > or = 220 kD Fr > 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 microM, and their IC50s were 13-39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1-loop fragment is important for optimal molecular interaction with GPIb alpha under high shear stress.     

Thrombopoietin and platelet aggregation in patients with stable coronary artery disease

Thrombopoietin (TPO) may facilitate platelet activation and aggregation. However, data on the impact of TPO on platelet aggregation in patients with stable coronary artery disease (CAD) are scarce. We aimed to investigate associations between TPO and platelet aggregation and activation in patients with stable coronary artery disease (CAD). We studied 900 stable CAD patients. Serum TPO was assessed by ELISA. Platelet aggregation was evaluated using the Multiplate Analyzer (agonists: arachidonic acid [AA] and collagen) and the VerifyNow Aspirin Assay. Platelet activation was evaluated by soluble (s)P-selectin. Cyclooxygenase-1 inhibition was evaluated by serum thromboxane B₂ (TXB₂). We found that TPO correlated weakly with platelet aggregation evaluated by Multiplate using AA (r = ?0.09, p = 0.01) and collagen as agonists (r = ?0.03, p = 0.43) and by VerifyNow (r = 0.07, p = 0.03). We found no correlation between TPO and sP-selectin (r = ?0.01, p = 0.70). Independent predictors of AA-induced platelet aggregation by Multiplate included high levels of sP-selectin and serum TXB₂, high platelet count, increasing age and body mass index, female sex, and active smoking. Independent predictors of TPO included low AA-induced platelet aggregation by Multiplate, high levels of hs-CRP, active smoking, and high platelet aggregation evaluated by VerifyNow. In conclusion, TPO levels did not correlate with platelet activation and only weak associations were found between TPO and platelet aggregation, suggesting that TPO did not substantially facilitate platelet aggregation in stable CAD patients.     

Sudden death induced by intracoronary platelet aggregation

Sodium arachidonate (AA, 1.5 mg/kg) was injected into the coronary arteries in 16 rabbits. Arrhythmia, marked ST-T depression and apnea appeared in all cases, and 7 cases died within 10 min after the AA. Before the injection, the thromboxane B2 (TXB2) value was 1.2 +/- 0.2 ng/ml (mean +/- SE) and the 6-keto-PGF1 alpha value was 2.1 +/- 0.4 ng/ml. Three minutes after the AA, TXB2 values were 5.0 +/- 1.1 in the surviving cases and 17.9 +/- 6.5 ng/ml in the cases which died. 6-keto-PGF1 alpha values were 47.7 +/- 4.6 in the surviving cases and 248.5 +/- 69.3 ng/ml in the cases which died. There occurred no deaths in 7 cases pretreated with aspirin (ASA) and in 11 cases pretreated with OKY-046 and 1581, which are specific inhibitors of TXA2 synthetase. TXB2 values did not change in the ASA and OKY groups after the AA injection. 6-keto-PGF1 alpha values did not change in the ASA group and increased in the OKY group after the AA injection. Histological findings of the heart showed more remarkable ischemic changes in the non-pretreated group than in the ASA and OKY groups. These results suggest a role for TXA2 in sudden death. PGI2 production was extremely enhanced, suggesting the presence of a protective mechanism against thrombogenesis in vivo.     

High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.     

Thrombopoietin and platelet function

In hematopoietic stem cells and megakaryocytes, the thrombopoietin (TPO) receptor signals to control proliferation, maturation, and antiapoptosis. In the anucleated platelet, much of this signaling appears out of place. Nevertheless, platelets possess TPO receptors and the cascades for transduction of TPO signals, but the final effect has shifted from DNA regulation to control of platelet aggregation and secretion. Although at physiological concentrations, TPO is not a sufficiently strong stimulator to trigger platelet functions in the absence of a second stimulator, TPO increases the sensitivity of platelets to agonist stimulation, leading to better functions at lower stimulation.     

Factors affecting the observation of a synergistic response in platelet aggregation induced by pairs of excitatory agonists

When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 μM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).     

Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study

The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30–34°C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37°C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate® at five different test temperatures between 30°C and 34°C. Aggregation responses at 37°C served as controls. At temperatures of mild and moderate hypothermia (30–34°C), overall platelet aggregation was increased compared to 37°C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31°C and 34°C and in response to adenosine diphosphate between 30°C and 34°C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30°C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30°C and 34°C requires further detailed study.     

Thrombopoietin is synergistic with other hematopoietic growth factors and physiologic platelet agonists for platelet activation in vitro

Thrombopoietin (TPO) is the primary physiologic regulator of platelet production. The effect of TPO on platelet function, both alone and in combination with other hematopoietic growth factors, adenosine diphosphate (ADP), and epinephrine, was investigated using fluorescent-labeled antibodies to the activation-dependent antigen CD62 (P-selectin) and flow cytometry. TPO stimulated CD62 expression on normal human platelets, and this expression was completely inhibited by the soluble extracellular domain of the TPO receptor, MPL. The growth factors granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO), but not interleukin-3 (IL-3) or stem-cell factor (SCF), also stimulated platelet activation. The combination of EPO, SCF, ADP, and epinephrine with TPO were synergistic for platelet CD62 expression. These data further support a role for TPO in modulating platelet function. Am. J. Hematol. 54:225–232, 1997 © 1997 Wiley-Liss, Inc.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.     

Reduction of platelet aggregation induced by euglycaemic insulin clamp

Summary To examine the effect of serum insulin independent of the level of blood glucose in vivo on platelet aggregation in healthy individuals, a euglycaemic insulin clamp was applied up to 4 h. During the clamp, blood glucose at 5.0 mmol/l and insulin levels at 100 U/ml were maintained. Blood samples were drawn before, 2 and 4 h after the start of the insulin clamp. The platelet aggregation induced by 1 mol/l and 2 mol/l ADP, 1 g/ml collagen and 2.7 mol/l epinephrine was measured in the blood samples. Platelet aggregation induced by adenosine diphosphate, collagen and epinephrine in the 4 h sample was significantly reduced from the pre-clamp value of 8.4% to 3.9% (p<0.05), 26.2% to 7.0% (p<0.01) and 31.8% to 9.1% (p<0.01), respectively. On the other hand, when the same individuals were infused with physiological saline and blood glucose (4.4 mmol/l) and insulin level (10 mIU/l) were kept within normal values, there was no difference between the values of induced platelet aggregation in samples drawn before and during the insulin infusion. It was concluded that hyperinsulinaemia reduces platelet aggregation in vivo when euglycaemia was maintained.     

Cyclooxygenase-2 inhibitors enhance shear stress-induced platelet aggregation.

OBJECTIVES: 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. BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.