Shear Stress-induced Platelet Activation 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.

Role of thrombin to non-physiological shear stress induced platelet activation and function alternation

ObjectiveNon-physiological shear stress (NPSS) and thrombin have two distinct mechanisms for activating platelets. NPSS in mechanically assisted circulation (MAC) devices can cause platelet dysfunction, e.g., by shedding its key receptors. In addition, patients with heart failure have increased levels of thrombin generation, which may further affect the NPSS-induced platelet dysfunction, resulting in device-associated complications. This study aimed to assess the combined effect of NPSS and thrombin in platelet activation, expression of adhesion receptors on the platelet surface, and alterations of platelet aggregation. MethodsFresh human blood from healthy donors was divided into two groups; one group was treated by adding 0.01 U/mL thrombin, and another group not treated with thrombin served as a control comparison. They were then pumped through a novel blood shearing device which produces similar shear stress conditions to those in the MAC devices. Three levels of NPSS (i.e., 75, 125, and 175 Pa) with a 1.0 s exposure time were selected for the shearing conditions. Expression of platelet activation markers (PAC-1, activated GPIIb/IIIa and CD62P, platelet surface P-selectin) were investigated along with the shedding of platelet receptors (GPIb, GPIIb/IIIa, and GPVI), generation of platelet microparticles, and Phosphatidylserine (PS)-positive platelets detected by flow cytometry. Platelet aggregation (induced by collagen/ristocetin) was measured by Lumi-aggregometry. ResultsPlatelet receptors were shed after exposure to NPSS showing a positive correlation with the level of shear stress. The generation of platelet microparticles and PS-positive platelets also increased with greater NPSS. Elevated NPSS decreased the platelet aggregation capacity. Platelet activation level increased with greater NPSS. Being treated by thrombin can further exacerbate these characteristics under same level of NPSS, except that platelet activation level drastically dropped after the exposure to 175 Pa NPSS in the thrombin-treated blood. ConclusionAfter being treated by thrombin, platelets became more susceptible to NPSS, resulting in more receptor shedding, platelet microparticles, and PS-positive platelets, thus limiting platelet aggregation capacity after exposure to NPSS. Platelet activation, in terms of PAC-1 and P-selectin, is an interim status competing between the expression and shedding of these makers/receptors. When platelets have reached a saturation level of activation, exposure to excessive NPSS can potentially impair activation.

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.

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.

Validation of a procedure to assess ASA-response in patients with decreased, initial TRAP induced aggregation

Whole blood aggregometry on the is a simple, fast and standardized method and it is widely used to assess platelet function under antiplatelet therapy. Reference ranges and a cut-off value as a measure of ASA response were established by measuring arachidonic acid induced aggregation (ASPI-test) in healthy volunteers and cardiac patients after and used to classify patients as ASA responders or non-responders. However, assessing the platelet function is highly affected by pre-analytical and analytical conditions and often reduced aggregation by TRAP induced aggregation (TRAP-test) is seen, rendering the samples difficult for interpretation of the ASPI-test and the responder status to ASA. We hypothesised that in this simplified model any preanalytical factor has the same effect on TRAP-testing as on ASPI-testing and that by calculating the ratio between a defined, normal TRAP-test result and the TRAP-test result measured for the individual patient this ratio could be applied to the measured ASPI-test thereby reaching a more valid discrimination between ASA responders and -non-responders. TRAP- and ASPI-test were performed in blood from ASA-treated volunteers and controls on Multiplate™ before an after pneumatic tube delivery as a model to stimulate shear stress induced platelet activation and aggregation. The calculated, normalised ASPI test result after tube delivery did not differ significantly from the initial ASPI test result although tube delivery had a significant impact on the measured ASPI test result. If applied to patients samples a definite judgement on the ASA response status of patients with reduced “general platelet activatability” could be given. Normalisation of the ASPI-test result using the TRAP-test result may provide a method to judge on the ASA response status in patients with decreased initial “general platelet activatability”.

Hyperglycemia-Induced Platelet Activation in Type 2 Diabetes Is Resistant to Aspirin but Not to a Nitric Oxide–Donating Agent

OBJECTIVEAcute, short-term hyperglycemia enhances high shear stress–induced platelet activation in type 2 diabetes. Several observations suggest that platelets in type 2 diabetes are resistant to inhibition by aspirin. Our aim was to assess comparatively the effect of aspirin, a nitric oxide–donating agent (NCX 4016), their combination, or placebo on platelet activation induced by acute hyperglycemia in type 2 diabetes.RESEARCH DESIGN AND METHODSIn a double-blind, placebo-controlled, randomized trial, 40 type 2 diabetic patients were allocated to 100 mg aspirin once daily, 800 mg NCX 4016 b.i.d., both of them, or placebo for 15 days. On day 15, 1 h after the morning dose, a 4-h hyperglycemic clamp (plasma glucose 13.9 mmol/l) was performed, and blood samples were collected before and immediately after it for platelet activation and cyclooxygenase-1 (COX-1) inhibition studies.RESULTSAcute hyperglycemia enhanced shear stress–induced platelet activation in placebo-treated patients (basal closure time 63 ± 7.1 s, after hyperglycemia 49.5 ± 1.4 s, −13.5 ± 6.3 s, P < 0.048). Pretreatment with aspirin, despite full inhibition of platelet COX-1, did not prevent it (−12.7 ± 6.9 s, NS vs. placebo). On the contrary, pretreatment with the NO donor NCX 4016, alone or in combination with aspirin, suppressed platelet activation induced by acute hyperglycemia (NCX 4016 +10.5 ± 8.3 s; NCX 4016 plus aspirin: +12.0 ± 10.7 s, P < 0.05 vs. placebo for both). Other parameters of shear stress–dependent platelet activation were also more inhibited by NCX 4016 than by aspirin, despite lesser inhibition of COX-1.CONCLUSIONSAcute hyperglycemia-induced enhancement of platelet activation is resistant to aspirin; a NO-donating agent suppresses it. Therapeutic approaches aiming at a wider platelet inhibitory action than that exerted by aspirin may prove useful in patients with type 2 diabetes.

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.

Effect of doxazosin gastrointestinal therapeutic system on platelet degranulation and platelet–leukocyte microaggregate formation induced by physiologic shear stress in hypertension

Introduction In this prospective, ex vivo, single-blind study, the effect of doxazosin on platelet function was studied in patients with hypertension. Materials and methods Platelet activation by shear stress was measured in whole blood samples of 22 hypertensive patients and 22 normotensive controls, using flow cytometry. Sheared samples were evaluated for CD62 expression, microaggregate formation, and Ca 2+ mobilization. Results were collected at baseline and after 1 and 2 months of single-dose (4 mg/d) extended-release doxazosin gastrointestinal therapeutic system therapy. Results Doxazosin normalized blood pressure in hypertensive patients after 1 and 2 months of treatment. Hypertensive patients had a higher baseline percentage (mean ± SD) of degranulated platelets (CD62+) than the normotensive control group (4.14 ± 1.05 vs. 2.47 ± 0.68, P < 0.01). After 2 months of doxazosin gastrointestinal therapeutic system treatment, the percentage of CD62+ in the experimental group significantly decreased ( P < 0.05). At baseline, the number of platelet–leukocyte aggregates in vivo was greater in hypertensive patients ( P < 0.01); doxazosin did not normalize this measurement. Following shearing, platelet expression of CD62 increased significantly in the hypertensive group ( P < 0.001 vs. control). Shear stress-induced platelet activation and microaggregate formation were also greater in hypertensive patients. Intraplatelet-free calcium concentration was higher in hypertensive patients at baseline than in the normotensive group ( P < 0.001). At 2 months, doxazosin significantly reduced thrombin-stimulated Ca 2+ mobilization in hypertensive patients ( P < 0.01 vs. baseline). Conclusions Platelets from hypertensive patients are more readily activated by shear stress and demonstrate significant alterations in cytoplasmic-free calcium mobilization. Doxazosin treatment reduced blood pressure and normalized alterations in platelet function.

Targeting shear stress-induced platelet activation: is lesion-specific antiplatelet therapy a realistic clinical goal?

Platelets are mediators of physiologic hemostasis and pathologic thrombosis. They operate within distinctive vascular and rheologic microenvironments, and their participation in hemostasis or thrombosis is directed by distinct variables operating within the microenvironment. Thrombosis is not simply too much hemostasis: there is good evidence that triggering mechanisms of platelet aggregation under low shear stress conditions are different from those operating under high shear stress conditions. Such differences are hypothesized to exist in vivo and to separate mechanisms of microvascular hemostasis from mechanisms of arterial thrombosis, such as those involved in myocardial, cerebral and peripheral vascular ischemia and infarction. This separation forms the conceptual basis for the hypothesis that lesion-specific antithrombotic agents might some day be invented that inhibit arterial thrombosis without causing bleeding that arises from impaired hemostasis. The focus of much of the work in this field has been platelet aggregation initiated by shear-dependent von Willebrand factor binding to the platelet glycoprotein Ib-IX-V complex. It is hypothesized that by elucidating molecular mechanisms of platelet activation operating under pathologically elevated shear stresses, targets of lesion-specific therapies will one day be identified for use in clinical syndromes of arterial thrombosis.

Acute, short-term hyperglycemia enhances shear stress-induced platelet activation in patients with type II diabetes mellitus

ObjectivesThe aim of our study was to assess whether acute, short-term hyperglycemia affects platelet reactivity in patients with Type II diabetes mellitus (T2DM). BackgroundHyperglycemic spikes are thought to precipitate ischemic events in T2DM. Previous studies have shown in vivo platelet activation in diabetes; however, no studies have assessed whether acute in vivo hyperglycemia induces further activation of platelets. MethodsIn a cross-over, randomized, double-blind study, 12 patients with T2DM underwent 4 h of either acute hyperglycemia (13.9 mmol/l, 250 mg/dl) or euglycemia (5.5 mmol/l, 100 mg/dl). Shear stress-induced platelet activation, P-selectin and lysosomal integral membrane protein (LIMP) expression on platelets in the bleeding-time blood, urinary 11-dehydro-thromboxane B2(TxB2) excretion, von Willebrand factor:antigen (vWF:Ag), and von Willebrand factor:activity (vWF:activity) were measured before and after hyperglycemia or euglycemia. ResultsShear stress-induced platelet activation, P-selectin and LIMP expression on platelets in the bleeding-time blood, and urinary 11-dehydro-TxB2excretion increased significantly after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. Plasma vWF:Ag and vWF:activity increased strikingly in parallel fashion after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. ConclusionsOur data demonstrate that acute, short-term hyperglycemia induces an increased activation of platelets exposed to high shear stress conditions in vitro (filtration method) or in vivo (bleeding time). In vivo platelet activation is reflected by an increased urinary excretion of 11-dehydro-TxB2. The increased levels of vWF in the circulation correlate with the increase in platelet activation markers and may indicate some degree of causation. Acute, short-term hyperglycemia in T2DM may precipitate vascular occlusions by facilitating platelet activation.

Shear stress-induced myosin association with cytoskeleton and phosphorylation in human platelets.

Upon platelet activation by a high shear stress (108 dyne/cm2), actin and actin-binding protein increased rapidly into the Triton-insoluble cytoskeleton, whereas the association of myosin increased gradually. The amounts of cytoskeleton-associated myosin depended on the extent of aggregation. Preceding the maximal aggregation and ATP secretion, the 20 kDa light chain of myosin (MLC) is rapidly phosphorylated to approx. 45% of 20 kDa MLC and is then dephosphorylated. Cytoskeletal association of myosin and phosphorylation of 20 kDa MLC was inhibited by OP-41483, a prostaglandin I2 analog, which inhibited the full aggregation response to shear stress. Exposure to high shear stress resulted in an increased association of myosin light chain kinase and protein phosphatase types 1 and 2A with the cytoskeleton, while the cytoskeletal association of protein kinase C was not evident. These results indicate that 20 kDa MLC phosphorylation is involved in shear stress-induced platelet activation, and that cytoskeletal association of protein phosphatases may regulate the phosphorylation level of cytoskeletal elements such as myosin together with myosin light chain kinase.

Inhibition of shear stress-induced platelet activation by commercial luminescence reagents

Inhibition of shear stress-induced platelet activation by commercial luminescence reagents