Shear-dependent Platelet Aggregation Research Articles

Shear-dependent platelet aggregation size.

Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet‐poor plasma (containing von Willebrand factor, vWF) and fluorochrome‐labeled platelets were subjected to a range of shear rates (0‐10 000 s−1) for 0, 5, 10, and 15 minutes using a custom‐built blood‐shearing device. Formed sizes of platelet aggregates under a range of shear‐controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13—ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s−1). We found that the average size of platelet aggregates formed under physiological shear rates of 360‐3000 s−1 (200‐300 μm2) was significantly larger compared to those sheared at >6000 s−1 (50‐100 μm2). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s−1 in order to maintain the optimal size of platelet aggregates to “plug off” the injury site and stop bleeding.

Shear-Dependent Platelet Aggregation: Mechanisms and Therapeutic Opportunities.

Cardiovascular diseases (CVD) are the number one cause of morbidity and death worldwide. As estimated by the WHO, the global death rate from CVD is 31% wherein, a staggering 85% results from stroke and myocardial infarction. Platelets, one of the key components of thrombi, have been well-investigated over decades for their pivotal role in thrombus development in healthy as well as diseased blood vessels. In hemostasis, when a vascular injury occurs, circulating platelets are arrested at the site of damage, where they are activated and aggregate to form hemostatic thrombi, thus preventing further bleeding. However, in thrombosis, pathological activation of platelets occurs, leading to uncontrolled growth of a thrombus, which in turn can occlude the blood vessel or embolize, causing downstream ischemic events. The molecular processes causing pathological thrombus development are in large similar to the processes controlling physiological thrombus formation. The biggest challenge of anti-thrombotics and anti-platelet therapeutics has been to decouple the pathological platelet response from the physiological one. Currently, marketed anti-platelet drugs are associated with major bleeding complications for this exact reason; they are not effective in targeting pathological thrombi without interfering with normal hemostasis. Recent studies have emphasized the importance of shear forces generated from blood flow, that primarily drive platelet activation and aggregation in thrombosis. Local shear stresses in obstructed blood vessels can be higher by up to two orders of magnitude as compared to healthy vessels. Leveraging abnormal shear forces in the thrombus microenvironment may allow to differentiate between thrombosis and hemostasis and develop shear-selective anti-platelet therapies. In this review, we discuss the influence of shear forces on thrombosis and the underlying mechanisms of shear-induced platelet activation. Later, we summarize the therapeutic approaches to target shear-sensitive platelet activation and pathological thrombus growth, with a particular focus on the shear-sensitive protein von Willebrand Factor (VWF). Inhibition of shear-specific platelet aggregation and targeted drug delivery may prove to be much safer and efficacious approaches over current state-of-the-art antithrombotic drugs in the treatment of cardiovascular diseases.

Von Willebrand Factor-ADAMTS13 Interactions

Abstract SCI-18ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) is a plasma metalloenzyme that cleaves von Willebrand factor (vWF), thereby limiting excessive platelet adhesion and aggregation and attenuating the rate of thrombus formation at the site of injury. Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome, while mild to moderate deficiency of plasma ADAMTS13 activity is associated with an increased risk for myocardial infarction and stroke. Adamts13 null mice in the ApoE null background exhibit a dramatically increased systemic inflammation and early atherosclerosis. Thus, ADAMTS13 plays a crucial role in pathogenesis of arterial thrombotic disorders such as TTP and chronic inflammatory diseases such as atherosclerosis. vWF is the only known substrate for ADAMTS13 to date. Structure-function analysis demonstrates that the N-terminal portion of ADAMTS13 (MDTCS) is sufficient for proteolytic cleavage of vWF, while the C-terminal portion of ADAMTS13 (T2–8 and CUB) inhibits shear-dependent platelet aggregation via free thiols (-SH). The proteolytic cleavage of vWF by ADAMTS13 is dramatically accelerated by binding of Factor VIII (FVIII) and/or platelets under shear stress. A B-domainless FVIII (FVIII-SQ) or a light chain of FVIII (FVIII-LC) appears to be sufficient for the enhanced proteolysis of vWF by ADAMTS13 in vitro and in vivo using a murine model. In contrast, a heavy chain of FVIII (FVIII-HC) or a light chain lacking an acidic region (a3) (FVIII-LC#x2610;¢a3) exhibits no rate enhancing effect under the same conditions. These results suggest that the light chain, particularly the acidic (a3) region that contains a high-affinity vWF binding site is required for the cofactor activity in accelerating vWF proteolysis. Consistent with this hypothesis, mutations in the D'D3 domain of vWF naturally occurring in patients with type 2N von Willebrand disease that reduces FVIII binding also impair the FVIII-dependent proteolysis by ADAMTS13 under fluid shear stress. To develop novel therapeutics, we have constructed, expressed, and screened for a panel of ADAMTS13 variants with a modification of the exosites (motif A and motif B) in the spacer domain. We identified several ADAMTS13 variants that have enhanced specific activity and are also resistant to inhibition by autoantibodies against ADAMTS13 in patients with acquired idiopathic TTP. These gain-of-function and autoantibody-resistant ADAMTS13 variants may be further developed as potential therapeutics for acquired TTP with inhibitors. Disclosures:No relevant conflicts of interest to declare.

Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation

AbstractDisturbances of blood flow at sites of atherosclerotic plaque rupture are one of the key pathogenic events promoting platelet activation and arterial thrombus formation. Shear effects of platelets have been extensively investigated in vitro; however, the mechanisms by which shear promotes platelet aggregation in vivo remain incompletely understood. By employing high-resolution imaging techniques to in vitro and in vivo thrombosis models, we demonstrate a unique mechanism initiating shear-dependent platelet aggregation involving aggregate formation between discoid platelets. These discoid platelet aggregates are initially unstable and result from the development of membrane tethers between coadhering platelets. Tether formation involves the adhesive function of GPIb/V/IX and integrin αIIbβ3, and conversion of discoid platelet aggregates into stable aggregates requires released ADP. The efficiency of this process is regulated by 3 independent variables, including the reactivity of the adhesive substrate, the level of shear flow, and the platelet density at the adhesive surface. These studies identify a new mechanism initiating platelet aggregation that is critically influenced by shear, physical proximity between translocating platelets, and membrane tether formation. Moreover, they provide a model to explain how the discoid morphology of platelets facilitates the maintenance of adhesive interactions with thrombogenic surfaces under high shear stress conditions.

Role of the Pyk2–MAP Kinase–cPLA2Signaling Pathway in Shear-Dependent Platelet Aggregation

Mechanisms of shear-induced platelet aggregation are not established. Data that ristocetin-induced von Willebrand factor (VWF) binding to glycoprotein (Gp) Ibalpha activates proline-rich tyrosine kinase 2 (Pyk2) and extracellular-regulated kinase (ERK) has led to speculation that these events are coupled and that a MAP kinase may activate cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release. To test this hypothesis and clarify the role of AA metabolism in shear-induced VWF-dependent platelet aggregation, we examined Pyk2, ERK1/2, and p38 phosphorylation, and arachidonic acid release and metabolism in platelets subjected to pathological shear stress in vitro. We observe tyrosine phosphorylation of Pyk2, p38, and ERK1/2 but no measurable increase in free AA, 12-hydroxyeicosatetraenoic acid, or thromboxane A2. Inhibitors of ERK, p38, or cyclooxygenase activation fail to affect shear-induced platelet aggregation. When washed platelets are aspirin-pretreated, arachidonic acid release becomes measurable and aggregation at 60 and 120 s is attenuated. These data indicate that shear-induced VWF binding to platelet GpIb-IX-V activates Pyk2, ERK1/2, p38, and cPLA2, but that the magnitude of these responses is below the threshold needed to enhance shear-induced VWF-dependent platelet aggregation in the presence of plasma. These results provide a mechanistic basis for the long-standing observation that shear-dependent platelet aggregation is unaffected by the antiplatelet drug aspirin.

Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions

AbstractUnder conditions of high shear stress, both hemostasis and thrombosis are initiated by the interaction of the platelet membrane glycoprotein (GP) Ib-IX-V complex with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix or plasma. This interaction involves the A1 domain of vWF and the N-terminal extracellular region of GP Ibα (His-1-Glu-282), and it can also be induced under static conditions by the modulators ristocetin and botrocetin. In this study, a panel of anti-vWF and anti-GP Ibα antibodies—previously characterized for their effects on ristocetin- and botrocetin-dependent vWF–GP Ib-IX-V interactions—was analyzed for their capacity to inhibit either the adhesion of Chinese hamster ovary cells expressing recombinant GP Ibα to surface-associated vWF under hydrodynamic flow or shear-stress–induced platelet aggregation. The combined results suggest that the shear-dependent interactions between vWF and GP Ibα closely correlate with ristocetin- rather than botrocetin-dependent binding under static conditions and that certain anti-vWF monoclonal antibodies are able to selectively inhibit shear-dependent platelet aggregation.

Use of a platelet filter test in patients with thrombocytosis

In the differential diagnosis of primary and secondary thrombocytosis platelet function tests may play an important role. We examined the applicability of a platelet filter test (shear-dependent platelet aggregation) as a tool, to differentiate primary thrombocytosis (cases with myeloproliferative disorders) from secondary (reactive) thrombocytosis. The test was carried out in 53 patients suffering from myeloproliferative disorders associated with primary thrombocytosis and in 21 patients with other diseases complicated by secondary thrombocytosis. Using citrate as anticoagulant, the sensitivity of the O'Brien's test proved to be 77.1%, and its specificity 94.4%. Using heparin as anticoagulant the sensitivity and specificity of the test were found to be also reliably high. Based on these studies we suggest the use of the O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated platelet count. In the case of normal results, the causes of reactive thrombocytosis should be clarified first, while with abnormal results, haematological examination of the patients should be performed.

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

AbstractShear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Studies of the platelet filter test (shear dependent platelet aggregation) in patients with uncommon haemorrhagic disorders.

Platelets of anticoagulated whole blood forced at 40 mmHg through a fine filter are activated, aggregated and retained, so block the filter (platelet filter test, O'Brien JR, Salmon GP. Blood 1987; 1354-1361). Our clinical experiences with this simple and quick haemostasis test are summarized. Patients were investigated with different types of vWD (type-1 = 35, type-2A = 7, type-2B = 7, type-3 = 1), Glanzmann's thrombasthenia, congenital deficiency of cyclo-oxygenase, acquired Bernard-Soulier syndrome, FXII-, FXIII-deficiency and a control group. The cumulative drop count and the platelet retention were carefully measured during two phases of the filter test. Platelet count, bleeding time, vWF:Ag and vWF:Rcof activity were measured along with the platelet filter test. The filter was not blocked and the platelet retention was abnormally low in all patients with thrombasthenia, type-2a, type-2B, type-3 vWD. Treatment with 1-desamino-8-D-arginine-vasopressin (DDAVP) caused enhanced platelet retention in 16 patients with type-1 vWD. The test is simple, quick and cheap, has good reproducibility, and may be useful in clinical haemostasis laboratories for examination of high shear induced platelet functions.

Study of shear-dependent platelet aggregation in patients with low and high von Willebrand factor plasmatic level

Study of shear-dependent platelet aggregation in patients with low and high von Willebrand factor plasmatic level