Occlusive Platelet Thrombi Research Articles

A Large Cluster of Micrornas At 14q32 Defines an RNA Expression Module That Accounts for Racial Differences in Protease Activated Receptor 4-Mediated Platelet Reactivity

AbstractAbstract 380Myocardial infarction and stroke typically result from an occlusive platelet thrombus formed at the site of atherosclerotic plaques. There is reproducible variation in platelet reactivity among different individuals – a variation that likely contributes to thrombosis risk. The inter-individual variation in platelet reactivity is highly heritable, and this heritability is greater in African Americans (AAs) than European Americans (EAs), but there is limited understanding of the genetic mechanisms responsible for this variability. The Platelet RNA And eXpression-1 (PRAX1) study was designed to identify novel genes and miRNAs responsible for inter-individual variation in platelet reactivity. We assessed platelet aggregation in 70 AA and 84 EA healthy subjects and profiled miRNA and mRNA from highly purified blood platelets. The richness of these data provided unique opportunities to establish miRNA-mRNA-physiology relationships that led to a number of novel and unexpected results.Although 607 miRNAs were detected above background, some were virtually absent in some samples. Using highly stringent criteria based on a biphasic distribution of expression in the 154 samples, we defined 178 platelet miRNAs present in at least 65% of subjects. Statistical analysis identified miRNAs that were differentially expressed (DE) by age, gender and race. A miRNA-miRNA correlation matrix uncovered a cluster of 52 miRNAs located at 14q32 that were highly co-regulated and that were expressed at higher levels in platelets from EAs than AAs. Unexpectedly, platelet aggregation in response to PAR4-AP was significantly higher in AAs than in EAs, whereas no racial difference was observed for the platelet aggregation response to arachidonic acid, ADP or PAR1-AP. Having established that 14q32 miRNAs were DE by race and that PAR4-mediated platelet reactivity was different by race, we sought to determine whether there were mRNAs that linked these two discoveries. Using a simple linear regression with an FDR q<.25, we found 119 mRNAs that were positively correlated with PAR4 reactivity. Notably, the gene encoding PAR4 (F2RL3) was not DE by race. A substantially higher proportion of 14q32 miRNAs were represented among the miRNAs enriched for targets among PAR4-positively-correlated mRNAs than would be expected by chance (p = 0.0092, OR=3.71). In other words, the racially-differentially expressed miRNAs located on 14q32 target mRNAs that are positively associated with PAR4 reactivity. A conditional correlation analysis showed that the significant association between PAR4 reactivity and race vanished after accounting for RNA expression, indicating that these two traits are highly collinear. We were especially intrigued with the gene, PCTP, which encodes phosphatidylcholine (PC) transfer protein (TP), because of the well-established importance of fatty acids, phospholipids and PC in platelet reactivity and because PC-TP catalyzes the transfer of PC between membranes. PCTP was DE by race (q value=10−19) and by PAR4 reactivity (p=10−6), and PC-TP protein was higher in AAs than EAs. PCTP is predicted to be targeted by 14q32 miRNA, hsa-miR-376c, and we validated that PC-TP mRNA and protein were knocked down by hsa-miR-376c.In summary, we have discovered racial differences in PAR4-mediated platelet activation and uncovered a genetic regulatory module in which racial differences in expression of miRNAs located at 14q32 and their target mRNAs account for this racial difference in platelet function. High levels of 14q32 miRNAs in EAs specifically target mRNAs that positively regulate PAR4 activity, resulting in lower sensitivity to PAR4-mediated activation. The inverse is true of AAs. Compared to EAs, AAs have a 2-fold increase in the incidence of CHD and a lower long-term survival. In addition, it is unknown whether the racial difference in platelet reactivity impacts the benefits and risks of anti-platelet therapies. Because novel inhibitors of PAR1 and 4 are currently in clinical development, it is critical to know whether dosing of such agents should be adjusted by race to maximize benefit and avoid toxicity. Lastly, since PAR4 is expressed in tissues other than platelets (e.g., heart, brain, liver), if 14q32 miRNA targets are DE by race in non-platelet tissues, it is possible that the consequent altered gene expression could contribute to non-thrombotic diseases known to show racial differences. Disclosures:No relevant conflicts of interest to declare.

Impaired thrombolysis: a novel cardiovascular risk factor in end-stage renal disease

End-stage renal disease (ESRD) patients have an excess cardiovascular risk, above that predicted by traditional risk factor models. Prothrombotic status may contribute to this increased risk. Global thrombotic status assessment, including measurement of occlusion time (OT) and thrombolytic status, may identify vulnerable patients. Our aim was to assess overall thrombotic status in ESRD and relate this to cardiovascular risk. Thrombotic and thrombolytic status of ESRD patients (n = 216) on haemodialysis was assessed using the Global Thrombosis Test. This novel, near-patient test measures the time required to form (OT) and time required to lyse (lysis time, LT) an occlusive platelet thrombus. Patients were followed-up for 276 ± 166 days for major adverse cardiovascular events (MACE, composite of cardiovascular death, non-fatal MI, or stroke). Peripheral arterial or arterio-venous fistula thrombosis was a secondary endpoint. Occlusion time was reduced (491 ± 177 vs. 378 ± 96 s, P < 0.001) and endogenous thrombolysis was impaired (LT median 1820 vs.1053 s, P < 0.001) in ESRD compared with normal subjects. LT ≥ 3000 s occurred in 42% of ESRD patients, and none of the controls. Impaired endogenous thrombolysis (LT ≥ 3000 s) was strongly associated MACE (HR = 4.25, 95% CI = 1.58-11.46, P = 0.004), non-fatal MI and stroke (HR = 14.28, 95% CI = 1.86-109.90, P = 0.01), and peripheral thrombosis (HR = 9.08, 95% CI = 2.08-39.75, P = 0.003). No association was found between OT and MACE. Impaired endogenous thrombolysis is a novel risk factor in ESRD, strongly associated with cardiovascular events.

A Rare Case of Thrombotic Thrombocytopenic Purpura Presenting With Pancreatitis

Thrombotic thrombocytopenic purpura (TTP) is a rare disease characterized by platelet consumption in thromboses, leading to a microangiopathic hemolytic anemia (MAHA). Thrombotic thrombocytopenic purpura involves a decrease in the activity of a metalloproteinase, ADAMTS13, which is critical in regulation of the coagulation cascade. This occurs in several conditions, and therefore, TTP is associated with a number of diseases. The decreased activity of ADAMTS13 leads to occlusive platelet thrombi, which can result in a variety of tissue manifestations. Patients normally present with nonspecific symptoms, such as fever and petechia. The classic pentad, although now extraordinarily uncommon, includes thrombocytopenia, MAHA, varying degrees of renal failure, fever, and nonlocalizable neurologic symptoms. We present a rare case in which pancreatitis was the presenting condition in a patient with acute idiopathic TTP. We discuss the pathogenesis and etiologies of these 2 diseases.

Platelet-mediated erythromelalgic, cerebral, ocular and coronary microvascular ischemic and thrombotic manifestations in patients with essential thrombocythemia and polycythemia vera: A distinct aspirin-responsive and coumadin-resistant arterial thrombophilia

Microvascular circulation disturbances including erythromelalgia, its microvascular ischemic complications, and migraine-like atypical or typical transient ischemic cerebral, ocular, and coronary ischemic attacks are specific clinical manifestations in patients with essential thrombocythemia (ET), and polycythemia vera (PV) associated with thrombocythemia. Thrombocythemia (ET and PV) patients with microvascular disturbances have shortened platelet survival, increased β-thromboglobulin (β-tg), platelet factor 4 (PF4), and thrombomoduline (TM) levels, and increased urinary thromboxane B2 (TxB2) excretion indicating platelet-mediated processes in vivo. Inhibition of platelet cyclooxygenase (COX 1) by aspirin is followed by relief of microvascular disturbances, correction of shortened platelet survival, and return of plasma levels of β-tg, PF4, TM levels and TxB2 excretion to normal. The transient ischemic attacks and thrombotic complications in thrombocythemia are very likely caused by hypersensitive platelets produced by spontaneously proliferating enlarged megakaryocytes in the bone marrow of ET and PV patients. In contrast to normal platelets in healthy individuals the circulating hypersensitive thrombocythemic platelets spontaneously activate and secrete their products, thus forming aggregates that transiently plug the microcirculation, or result in occlusive platelet thrombi in arterioles or small arteries. Clear evidence is presented that the microvascular transient ischemic and occlusive thrombotic complications in thrombocythemia patients are relieved by treatment with aspirin and by reduction of platelet counts to normal (<400 × 109/l), but not by coumadin. In patients with thrombocythemia associated with PV, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular ischemic and thrombotic syndrome of thrombocythemia to major arterial and venous thrombotic complications. Correction of hematocrit and blood viscosity by phlebotomy significantly reduces the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in PV patients are obtained by treatment with low-dose aspirin on top of phlebotomy or by treatment with the platelet lowering agents, anagrelide, interferon or hydroxyurea.

Coronary artery intimal hyperplasia, occlusive platelet thrombus, and fatal myocardial infarction in an intravenous cocaine user

A 38-year-old man died of an acute inferoposterior myocardial infarction, shortly after intravenous injection of cocaine. At necropsy, diffuse intimal hyperplasia and an occlusive platelet thrombus involved the left main, left anterior descending, and the left circumflex coronary arteries. The possible relation of these morphologic findings to the effects of cocaine on platelet and coronary vascular function are discussed and the relevant literature is reviewed.

Thrombotic Thrombocytopenia Induced in Dogs and Pigs

Thrombotic thrombocytopenia with severe depletion of plasma von Willebrand factor (vWF) was induced in normal large animals (5 dogs and 2 pigs) by botrocetin, a Bothrops factor requiring vWF for platelet agglutination. Botrocetin (90 to 100 U/kg, 2.14 to 2.38 mg/kg, in a single i.v. injection) reduced plasma vWF activity to < 0.1 U/mL for 24 hours. During this period, multimeric analysis of plasma vWF antigen (Ag) revealed the loss of intermediate- and high-molecular-weight forms with a concomitant increase in lower molecular weight forms. A moderate reduction in factor VIII (FVIII) activity was observed. The vWF reduction was accompanied by transient thrombocytopenia and prolonged bleeding times during the deficiency state. Occlusive platelet thrombi were detected by transmission electron microscopy in the microcirculation of lung and spleen but not kidney or brain 30 minutes after the botrocetin injection. Recovery of plasma vWF and platelet count occurred within 48 hours and was associated with the appearance in the plasma of unusually large forms of vWF:Ag multimers. The vWF:Ag multimer distribution was normal at 72 hours. The ultrastructural distribution of vWF in unstimulated normal porcine and canine platelets was examined by using immunogold staining. VWF was detected in the alpha-granules of normal pig platelets but was not observed in platelets from normal dogs. However, both animals developed thrombotic thrombocytopenia when injected with botrocetin. A second group of animals (2 dogs and 3 pigs) with von Willebrand disease (vWD) was given a single botrocetin injection (90 to 100 U/kg). No thrombocytopenia occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxidation by activated platelets: A possible link between thrombosis and atherogenesis

Generation of reactive oxygen species (ROS) by platelets was detected by a cyano-tetrazolium dye (CTC) which forms fluorescent formazan on the cell surface upon reduction. Fluorescence was quantitated by a densitometric device. Resting platelets in plasma produced significant fluorescence ( P < 0.0001), and addition of thrombin enhanced the fluorescence of the coagulated platelet mass even further (by 2 h, a 6- and 8-fold increase over fluorescence of platelet-free plasma was measured, respectively). Blood containing CTC was perfused through a glass capillary tubing and the action of shear forces resulted in the formation of an occlusive platelet thrombus. Such thrombi (formed either from whole blood or platelet-rich plasma) were intensely fluorescent, indicating formation of ROS in the platelet mass (a 10- and 8-fold increase in fluorescence over coagulated plasma, respectively). Lipid peroxide content of resting platelets in platelet-rich plasma was doubled over 24 h storage, while addition of thrombin caused a 7.4-fold increase ( P < 0.0001) of lipid peroxides in the retracted platelet-rich plasma-clot. Transition metal chelator and antioxidant prevented lipid peroxidation by platelets in response to thrombin. Thrombin activation of (washed) platelets in plasma-free medium caused only 1.4-fold increase in oxidation of added low density lipoprotein (LDL). In contrast, thrombin activation of platelets suspended in de-lipidated autologous plasma resulted in a 5.25-fold increase ( P < 0.0001) in LDL oxidation. Generation of ROS and lipid peroxides by platelets can be an important mechanism through which thrombotic events contribute to atherogenesis.

Severe myocardial ischemia and impaired contractile function caused by acute occlusive platelet thrombi in stenosed coronary arteries

Severe myocardial ischemia and impaired contractile function caused by acute occlusive platelet thrombi in stenosed coronary arteries

The effects of cigarette smoke and nicotine on platelet thrombus formation in stenosed dog coronary arteries: inhibition with phentolamine.

This study was undertaken to examine in vivo the effects of cigarette smoke on cyclic reductions in coronary flow due to platelet thrombus formation in the stenosed coronary arteries of anesthetized dogs. The circumflex coronary artery of 21 mongrel dogs was stenosed 60 - 80%, with blood flow measured with an electromagnetic flow probe. After the administration of cigarette smoke, plasma epinephrine was elevated nine times the control level (p less than 0.001) and peak mean blood pressure was elevated one and one-half times control (p less than 0.01). The hematocrit increased several percent (p less than 0.01) with cigarette smoke, although blood gases and pH remained unchanged. In all 21 dogs, spontaneous reductions in coronary blood flow were greatly exacerbated in the stenosed circumflex artery as evidenced by the number of flow reductions, the increased size of the reductions and the rate of flow reduction. Nicotine administered intravenously in doses comparable to those achieved through absorption of cigarette smoke by the lungs provoked similar responses of alpha-adrenergic stimulation and potentiation of the platelet thrombus formation. An alpha-adrenergic antagonist, phentolamine, was given (3 mg/kg) intravenously to inhibit the exacerbated platelet thrombus formation due to cigarette smoke or infused nicotine. In 18 of 21 dogs, an acute occlusive platelet thrombus was prevented 15 minutes after phentolamine and after phentolamine and after a cigarette smoke or nicotine challenge. This study confirms a link between cigarette smoking, platelet formation, and the potential for humans to develop an acute occlusive platelet thrombus in a diseased and stenotic coronary artery.

Intravascular platelet aggregation in the heart induced by norepinephrine. Microscopic studies.

Aggregated platelets and occlusive platelet thrombi were found in small myocardial vessels of dogs on electron-microscope examination after prolonged infusion of norepinephrine. The etiology of the myocardial necrosis and fibrosis induced by catecholamines in experimental animals and seen in patients with pheochromocytoma and patients after norepinephrine treatment for shock may be related to this intravascular platelet-aggregating effect of catecholamines. The link between stress and acute myocardial infarction may be via catecholamine-induced intravascular platelet thrombosis. If the thrombogenic theory of atherosclerosis is valid, platelet aggregation induced by catecholamines may be the mechanism whereby arteriosclerotic heart disease is related to stress.