Platelet Hyperresponsiveness Research Articles

In Situ Microparticles Loaded with S-Nitrosoglutathione Protect from Stroke.

Treatment of stroke, especially during the first hours or days, is still lacking. S-nitrosoglutathione (GSNO), a cerebroprotective agent with short life time, may help if administered early with a sustain delivery while avoiding intensive reduction in blood pressure. We developed in situ forming implants (biocompatible biodegradable copolymer) and microparticles (same polymer and solvent emulsified with an external oily phase) of GSNO to lengthen its effects and allow cerebroprotection after a single subcutaneous administration to Wistar rats. Arterial pressure was recorded for 3 days (telemetry, n = 14), whole-blood platelet aggregation up to 13 days (aggregometry, n = 58), and neurological score, cerebral infarct size and edema volume for 2 days after obstruction of the middle cerebral artery by autologous blood clots (n = 30). GSNO-loaded formulations (30 mg/kg) induced a slighter and longer hypotension (-10 vs. -56 ± 6 mmHg mean arterial pressure, 18 h vs. 40 min) than free GSNO at the same dose. The change in pulse pressure (-50%) lasted even up to 42 h for microparticles. GSNO-loaded formulations (30 mg/kg) prevented the transient 24 h hyper-aggregability observed with free GSNO and 7.5 mg/kg-loaded formulations. When injected 2 h after stroke, GSNO-loaded microparticles (30 mg/kg) reduced neurological score at 24 (-62%) and 48 h (-75%) vs. empty microparticles and free GSNO 7.5 mg/kg and, compared to free GSNO, divided infarct size by 10 and edema volume by 8 at 48 h. Corresponding implants reduced infarct size and edema volume by 2.5 to 3 times. The longer (at least 2 days) but slight effects on arterial pressures show sustained delivery of GSNO-loaded formulations (30 mg/kg), which prevent transient platelet hyper-responsiveness and afford cerebroprotection against the consequences of stroke. In conclusion, in situ GSNO-loaded formulations are promising candidates for the treatment of stroke.

Platelet-Type von Willebrand Disease: New Insights into the Molecular Pathophysiology of a Unique Platelet Defect

Compared with coagulation factor defects, little attention is given to defects of platelet function as causes of rare bleeding disorders. Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant bleeding disorder and is unique among platelet disorders because it is characterized by platelet hyperresponsiveness rather than decreased function. The disease is caused by gain-of-function mutations in the platelet GP1BA gene, which codes for the platelet von Willebrand factor (VWF) receptor, GPIbα. Only five mutations (four missense and one deletion) have so far been reported. Affected patients suffer from mild to moderate mucocutaneous bleeding, low VWF activity compared with antigen, decreased high-molecular-weight VWF multimers, variable degree of thrombocytopenia and typically platelet aggregation in response to low concentrations of ristocetin. All reported PT-VWD missense mutations occur within the R-loop of GPIbα and it was speculated that the introduction of short branched chain mutations such as Val in PT-VWD stabilized the extended β-hairpin. Examination of this theory by surveying all the available GPIbα structures showed that a distinct conformation predominates for the R-loop when GPIbα is not bound to VWF-A1 and this provides the framework of a new hypothesis for the molecular basis of PT-VWD. Worldwide efforts to improve diagnosis of PT-VWD continue, and international systematic studies are required to further our understanding of the phenotype and the influence of the hyperresponsive GPIbα beyond hemostasis.

A novel assay of thrombotic risk

It is widely believed that platelet responsiveness can be used as a marker for assessing thrombotic risk in patients. Therefore, there have been many attempts to develop suitable assays for quantifying platelet responses in clinical samples 1. Platelet aggregometry is widely used, but is limited in its ability to assess platelet hyper-responsiveness. In order to develop a better assay of thrombotic risk, we use a novel assay to evaluate platelet secretion of adenosine triphosphate & diphosphate (ATP/ADP) in response to various agonists. This assay measures both the maximal amount of adenine nucleotides released by a range of platelet activators and the potency of each activator.

Toward a guided approach to platelet activation in diabetes

Diabetes is associated with substantially poorer cardiovascular outcomes. Furthermore, people with diabetes are at greatly increased risk of not responding to standard antiplatelet therapy for acute coronary syndromes, leading to higher mortality and stent thrombosis [1]. Poor glucose control likely contributes to these cardiovascular events through changes in platelet membrane fluidity and signalling, increased production of platelets leading to a greater fraction of younger and more reactive platelets not yet exposed to antiplatelet therapy, and increased triggering of platelet activation arising from an underlying thromboinflammatory milieu [2]. While patients with higher thrombotic risk may be prospectively identified through laboratory identification of elevated blood glucose or HbA1c [3], the approach to treatment for diabetic platelet hyper-responsiveness remains unclear. Options such as increasing frequency of antiplatelet therapy, additional and more aggressive antiplatelet therapy or more intensive glucose control have been suggested (Fig. 1). However, the data to support these are limited and, particularly where more aggressive antiplatelet therapy is proposed, must be measured against the potential for increased bleeding risk in patients with hyperglycemia. In patients with type-2 diabetes, Capodanno et al. [4] demonstrated enhanced inhibition of platelet function when patients took 81 mg of aspirin twice per day compared with 81 mg daily. Spectre et al. [5] showed greater inhibition of platelet function with 75 mg twice per day when compared to 75 mg daily in patients with type-2 diabetes and vascular complications. In a recent study Rocca et al. [6] showed that increasing frequency of dose better attenuated platelet activation over 24 h in both diabetic and non-diabetic patients. Therefore increasing the frequency of antiplatelet therapy dosing represents an appealing strategy in patients with diabetes and poor glycemic control. However, the clinical effectiveness of this approach on cardiovascular outcome, as well as the net benefit against increased bleeding risk remains to be investigated. Similarly, the addition of more aggressive antiplatelet therapy, such as newer ADP receptor antagonists with greater potency and bioavailability, or emerging thrombin receptor antagonists have been suggested [2]. The use of these must be weighed against the potentially increased risk of bleeding. While altering antiplatelet therapy strategies in patients with diabetes and poor glycemic control is an appealing avenue for further clinical investigation, a potential alternative or complimentary approach might be to achieve improved glycemic control in order to attenuate platelet activation. Several studies have assessed the ability of infused insulin in patients with diabetes to attenuate platelet function and improve cardiovascular outcome in acute coronary syndromes [7]. While evidence of improvement in cardiovascular outcome is variable, intensive treatment with insulin in patients with diabetes may attenuate platelet activation relative to conventional approaches to glycemic control [8]. In this issue of the Journal of Thrombosis and Thrombolysis, Vivas et al. [9] explore whether tight glycemic control with insulin infusion attenuates platelet reactivity in patients with an acute coronary syndrome and hyperglycemia at admission. In a post hoc analysis of the prospective, randomized, open-label CHIPs study, 67 patients with poor glycemic control (as measured by HbA1c [ 6.5 %) were M. D. Linden (&) Centre for Microscopy, Characterisation and Analysis, M510, University of Western Australia, 35 Stirling Highway, Nedlands, WA 6009, Australia e-mail: matthew.linden@uwa.edu.au

Lipid Oxidation and Cardiovascular Disease: Introduction to a Review Series

A therosclerosis and the associated adverse complications of cardiovascular disease are major causes of morbidity and mortality in people living a Western lifestyle. A role for excess cholesterol in the pathophysiology of atherosclerosis is clear. However, additional mechanisms driving the relevant pathological changes in a chronic disease such as atherosclerosis are those that constitute the acute inflammatory response.1 The essential elements of a physiological, and regulated, inflammatory response starts with stimulated endothelium,2 displaying adhesive molecules for circulating white blood cells. This is accompanied by localized production of cell type–specific agonists for adherent monocytes, neutrophils, or lymphocytes by the activated endothelium.3 These agonists then activate the migratory instruction set of adherent or rolling cells positioned to receive both adhesion- and agonist-related stimuli from activated vascular endothelial cells.4 Lipid oxidation products formed by virtually every vascular cell type participate in orchestrating these processes. We also have recently come to appreciate that the inflammatory process is actively limited by activation of a resolution phase, often via generation of structurally specific oxidized lipids whose function is to orchestrate resolution of inflammation.5 The overall goal of this review series is to place recent observations and insights in rapidly developing areas of lipid oxidation into the framework of cardiovascular disease. Enzymatic and free radical oxidation have prominent roles in cardiovascular disease through their oxidative modification of existing molecules. Lipids are primary targets of this modification because they are the primary repository of oxidizable olefinic or double bonds. Oxidized lipids are best understood as oxygenated arachidonic acid products, the prostaglandins, leukotrienes, and thromboxane A2, that have diverse and potent effects throughout the inflammatory and reparative responses. These oxidized lipid mediators are …

Deletion of the Semaphorin, Sema4D, but Not Inhibition of Sema4D Shedding by ADAM17, Impairs Platelet Function and Reduces Infarct Size After Myocardial Ischemia.

Abstract 771Introduction. Platelets are activated early during the reperfusion of ischemic myocardium, potentially exacerbating the extent of ischemia/reperfusion (I/R) injury. We have recently shown (Zhu, et al., PNAS 2007) that platelets express the semaphorin family member, sema4D, as do T-cells. Sema4D is a cell surface protein whose receptors are expressed by B-cells, monocytes and endothelial cells as well as platelets. Loss of sema4D expression in mice causes a defect in signaling downstream of the platelet collagen receptor, glycoprotein (GP) VI, inhibiting platelet function in vitro and in vivo, and reducing the extent of platelet hyperresponsiveness and atherothrombosis in the setting of dyslipidemia (Zhu, et al. ATVB 2009). Because of the role played by platelets, leukocytes and endothelial cells in reperfusion injury, here we asked whether loss of sema4D expression can also protect the heart, reducing the extent of damage following temporary ischemia. Methods. The left anterior descending coronary artery of anaesthetized mice was ligated for 45 min. After reperfusion for 48 hours, the mice were re-anesthetized and perfused with 2,3,5-triphenyltetrazolium chloride to measure the area of infarction. Fluorescent microspheres were used to delineate the area at risk. Comparisons were made between sema4D(−/−) and wild type mice produced by breeding sema4D(+/−) heterozygotes. Results. Although there was no difference between the sema4D(−/−) and WT mice with respect to either heart size or area at risk, we observed a substantial (57%) decrease in infarct size in the sema4D(−/−) mice expressed as a fraction of the area at risk (N=7-9, p<0.005). Since sema4D is shed from the surface of activated platelets and T-cells by the metalloprotease, ADAM17, producing a large bioactive fragment, we next asked whether the protection against ischemia/reperfusion injury conferred by the sema4D knockout is due to the loss of cell-associated or soluble sema4D. Chimeric mice were produced in which hematopoiesis was reconstituted in irradiated sema4D(+/+) mice using fetal liver cells from mouse embryos that lack functional ADAM17. This produces mice in which sema4D is expressed as usual in the hematopoietic lineages, but unable to be shed. Chimerism, inhibition of sema4D shedding and recovery of normal cell counts were confirmed after transplantation. The ischemia/reperfusion studies were repeated comparing chimeras reconstituted with ADAM17-deficient and ADAM17-replete fetal liver cells. In contrast to the sema4D knockout, the extent of infarction was the same whether or not ADAM17 was functional and sema4D was shed. Conclusions. Although the role of sema4D and its receptors have been studied most extensively in the context of T-cell interactions with B-cells, our previous studies have made a case for the involvement of sema4D in platelet:platelet and platelet:endothelial cell interactions. We now show for the first time that 1) loss of sema4D expression in mice confers protection against ischemia/reperfusion injury in the myocardium, and 2) preventing the formation of soluble, bioactive sema4D is insufficient to recapitulate this effect. Since sema4D and its receptors are expressed on more than just platelets, it cannot be concluded that the observed protection in the knockout is solely due to the absence of platelet sema4D. However, experience with other knockouts that reduce platelet function suggests that the defects that we have observed in sema4D(−/−) platelet function are likely to contribute. Regardless of whether expression on platelets is entirely or only partly responsible for the observed phenotype, sema4D is an interesting target for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

A cardiologist view of vascular disease in diabetes

Diabetes mellitus is a potent risk factor for the development of a wide spectrum of cardiovascular (CV) complications. The complex metabolic milieu accompanying diabetes alters blood rheology, the structure of arteries and disrupts the homeostatic functions of the endothelium. These changes act as the substrate for end-organ damage and the occurrence of CV events. In those who develop acute coronary syndromes, patients with diabetes are more likely to die, both in the acute phase and during follow-up. Patients with diabetes are also more likely to suffer from chronic cardiac failure, independently of the presence of large vessel disease, and also more likely to develop stroke, renal failure and peripheral vascular disease. Preventing vascular events is the primary goal of therapy. Optimal cardiac care for the patient with diabetes should focus on aggressive management of traditional CV risk factors to optimize blood glucose, lipid and blood pressure control. Targeting medical therapy to improve plaque stability and diminish platelet hyper-responsiveness reduces the frequency of events associated with atherosclerotic plaque burden. In patients with critical lesions, revascularization strategies, either percutaneous or surgical, will often be necessary to improve symptoms and prevent vascular events. Improved understanding of the vascular biology will be crucial for the development of new therapeutic agents to prevent CV events and improve outcomes in patients with diabetes.

Abstract 3477: Platelet Hyper-responsiveness in Aspirin-Treated Patients with Clinical Cardiovascular Disease Compared to Matched High Risk Controls

Background: Platelet hyper-responsiveness to activating stimuli during aspirin (ASA) therapy may discriminate between high risk subjects who have developed acute thrombotic cardiovascular disease (CVD) events (coronary disease and stroke) and those who are at increased risk but are disease free. We hypothesized that subjects with documented CVD would have greater platelet reactivity on ASA therapy compared to matched high risk non-CVD subjects. Methods: Subjects (N=228; 61 +/− 8 yrs, 69% male, 60% white) were selected from families with known CVD; 114 had prevalent CVD and were matched on age, sex, and race to 114 apparently healthy controls with risk factors but without clinical CVD. CVD risk factors were measured and therapy adherence was determined by questionnaires. Platelet reactivity on 81 mg ASA/day was determined by whole blood (WB) aggregometry, platelet function analyzer (PFA) closure time, thromboxane B2 (TxB2) release ex vivo , and urinary excretion of 11-dehyrothromboxane B2 (Tx-M) in vivo . Results. CVD cases had greater platelet reactivity by all measures, both unadjusted, and adjusted for age, sex, race and adherence (Table ). Multivariable adjustment for cardiac risk factors and statin therapy eliminated case-control differences for Tx-M, but not for the ex vivo measures of platelet activation. ASA therapy duration in CVD subjects (8.8 +/− 6.2 yrs) was not related to platelet function. Conclusions: Greater residual platelet reactivity exists during ASA therapy in CVD subjects compared to matched high risk controls, even controlling for CVD risk factors and adherence to therapy. The data suggest that platelet hyper-responsiveness during ASA chemoprophylaxis may differentiate patients with CVD from those who are at risk for CVD, but have not developed it. Platelet hyper-responsiveness may be an intrinsic property of CVD, related to as yet unidentified environmental or genetic factors.

Surface expression of fatty acid translocase (FAT/CD36) on platelets in myeloproliferative disorders and non-insulin dependent diabetes mellitus: effect on arachidonic acid uptake.

Increased platelet reactivity has been implicated in the vascular complications of myeloproliferative diseases and diabetes mellitus. The mechanisms of platelet hyperresponsiveness have not been fully explained. Expression of CD36 or fatty acid translocase (FAT) and its role in arachidonic acid (AA) uptake by platelets were examined in subjects with myeloproliferative disorders (MPD), those with non-insulin-dependent diabetes mellitus (NIDDM), and in normal, healthy, age-matched controls. Surface expression of CD36 on platelet membranes was increased in MPD (10.94 +/- 0.76 pmol/mg protein) compared with normal controls (6.94 +/- 0.48 pmol/mg protein), p < 0.001. Total platelet content of CD36 was also significantly higher (32.1 +/- 0.61 pmol/mg protein, p < 0.01) compared with those in sex and age matched normal controls (25.7 +/- 1.09 pmol/mg protein). In contrast, platelet surface expression of CD36 in NIDDM (6.5 +/- 0.56 pmol/mg protein) was not significantly different from those of normal controls despite higher total content of CD36 (32.8 +/- 1.2, pmol/mg protein, p < 0 .01). Intact MPD platelets bound significantly more arachidonic acid (AA) (1.53 +/- 0.16 nmol/mg protein, p < 0.05), compared with controls (1.12 +/- 0.07 nmol/mg protein) or NIDDM subjects (1.16 +/- 0.16 nmol/mg protein). The capacity of MPD platelet membranes to bind 14C-AA was also increased (1.72 +/- 0.25 nmol/mg protein, p < 0.05) compared with that of controls (1.62 +/- 0.05 nmol/mg protein) and of NIDDM (1.22 +/- 0.08 nmol/mg protein). This is consistent with higher surface expression of CD36 in MPD platelets. Membrane fatty acid analysis indicated that the % of AA in platelet phospholipids was significantly lower in MPD (3.15 +/- 0.81%) compared with the controls (5.62 +/- 1.7%, p < 0.05. The AA content of diabetic platelets (4.82 +/- 1.1%) was not significantly different from normal controls. In summary, both total and surface expression of CD36 are increased in MPD, consistent with an enhanced capacity for uptake of AA by platelets. Increased expression of CD36 in platelets may play a role in the vaso-occlusive manifestations of MPD.

Receptor dependent and -independent protein phosphorylation in platelets of spontaneously hypertensive rats

This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness in spontaneously hypertensive rats (SHR). For this purpose, 32P-labeled washed platelets of both SHR and normotensive controls Wistar-Kyoto (WKY) were challenged either with a receptor-mediated agonist (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between the two cell types. The data indicate that the exaggerated thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at least in part, to abnormalities in receptor-mediated transmembrane signalling.

Platelet phosphatidylcholine turnover in experimental hypertension.

To gain insight into the membrane alteration that could account for the hyperresponsiveness of platelets in hypertension, we have investigated whether, in resting platelets of hypertensive rats, the metabolism of phospholipids was modified. Because preliminary results indicated a specific acceleration of phosphatidylcholine turnover in spontaneously hypertensive rats, the possible relation between such an abnormality and hypertension was investigated by studying phosphorus-32 labeling of phosphatidylcholine (taken as an index of its turnover) in various experimental models of hypertension. The data showed that phosphatidylcholine turnover 1) was considerably increased in platelets from spontaneously hypertensive (even at the prehypertensive stage) and stroke-prone rats compared with Wistar or Wistar-Kyoto control rats, 2) did not differ between deoxycorticosterone-salt-treated hypertensive and control rats, and 3) was increased in Dahl salt-sensitive rats fed a high NaCl diet (hence hypertensive rats), compared with either the rats fed a low NaCl diet or the salt-resistant rats. These results indicate that an increase in phosphatidylcholine turnover is a consequence of neither hypertension nor high salt intake and appears likely to be of genetic origin. These data allow us to suggest the existence, in platelets, of a relation between phosphatidylcholine turnover, free cytoplasmic Ca2+, and responsiveness to stimuli. Because phosphatidylcholine is assumed to participate in signal transduction, an increase in its turnover in platelets might be considered as a primary membrane abnormality that, in primary hypertension, results in platelet hyperresponsiveness.

Attenuation of the inhibitory effect of prostacyclin on platelet function after tissue plasminogen activator or streptokinase infusion in the rabbit

The inhibitory effect of prostacylcin (PGI 2) on ex vivo platelet function was assessed after i.v. administration of tissue plasminogen activator (t-PA) or streptokinase (SK) to anaesthetised rabbits. Infusion of effective fibrinolytic doses of t-PA or SK caused marked enhancement of ex vivo platelet aggregation in response to collagen or arachidonic acid. This effect was most pronounced when subthreshold aggregating responses in the control period were convered to full, threshold, irreversible aggregating responses after fibrinolytic administration. Furthermore, the anti-aggregatory effect of PGI 2 on ex vivo platelet function was attenuated after infusion of t-PA or SK. Haemostatic and pharmacokinetic parameters were analysed in rabbits receiving t-PA infusion. Circulating plasma levels of plasminogen, fibrinogen and α2-antiplasmin were all decreased significantly and fibrinogen degradation products (FDP) were elevated significantly. To elucidate the mechanism(s) responsible for marked platelet hyperresponsiveness and loss of the inhibitory effect of PGI 2 after t-PA infusion, thromboxane B 2 (TxB 2) levels were measured in plasma from the same rabbits in which ex vivo platelet function was assessed. Fifteen min after t-PA infusion, TxB 2 levels were elevated significantly over control values. The findings of the present study revealed that fibrinolytic therapy induced a state of platelet hyperaggregability, with concomitant attenuation of PGI 2 activity on ex vivo platelet aggregation, as well as an elevation in circulating levels of thromboxane. As such, fibrinolytic-induced platelet activation may contribute to acute vascular reocclusion of recanalised coronary arteries after coronary thrombolysis and may thus limit the full therapeutic response to fibrinolytic agents.

Reduced platelet aggregability and increased vascular prostacyclin formation in a variant rat strain (IVA-SIV) with endogenous hypertriglyceridemia

The Ivanovas-Sieve (IVA-SIV) rat represents the only available animal model of endogenous hypertriglyceridemia, in the absence of obesity and/or overt diabetes. Since plasma lipids/lipoproteins can modulate platelet reactivity and eicosanoid metabolism, these were examined in two groups of Charles River (CR) and IVA-SIV rats of identical age. The IVA-SIV rats had 2-fold higher plasma triglycerides and a 55% higher number of circulating platelets; the number of platelets was significantly correlated with triglyceridemia. Platelet reactivity to ADP and to collagen was significantly reduced in these animals, whereas the formation of thromboxane B 2 did not differ from that of the CR. After perfusion of platelet-rich plasma (PRP) through the aortas of animals of the two strains, platelet aggregability, already lower in the IVA-SIV, was reduced to a higher extent compared to the CR. Increased levels of the prostacyclin metabolite 6-keto-PGF 1α were identified in the perfusate from the aortas of IVA-SIV rats. Platelets from these animals also showed an increased sensitivity to Iloprost, a stable prostacyclin analogue, with an IC 50 1.7-fold lower compared to CR rats. Spontaneous hypertriglyceridemia in the IVA-SIV model is not associated with platelet hyperresponsiveness, but rather with a reduced sensitivity to major aggregants.

Thrombin Binding and Response in Platelets From Patients With Dyslipoproteinemias: Increased Stimulus-Response Coupling in Type II Hyperlipoproteinemia

Platelets were obtained from patients with various hyperlipidemias [type II, type V, lecithin-cholesterol acyltransferase (LCAT) deficiency] and hypolipidemias (abetalipoproteinemia, Tangier disease) to ascertain relationships among plasma lipids, platelet lipids, thrombin binding and thrombin-induced platelet aggregation, and to compare these data with those previously obtained on stimulus-response coupling in platelets following in vitro modification of membrane microviscosity. Washed platelets were studied for their ability to bind 125I-thrombin in the range of 10(-10) to 10(-6) mol/L (10 mU/mL to 100 U/mL) and to aggregate with thrombin at concentrations less than 10(-9) mol/L (100 mU/mL). The values for binding and aggregation in eight patients from six kindred with familial hypercholesterolemia, taken as a group, fell in the low normal range. If divided into two groups, patients with overt cardiovascular disease bound normal amounts of thrombin but were more responsive to it, whereas patients without overt cardiovascular disease bound lower amounts of thrombin but gave an aggregation response in the normal range. These results suggest that platelet hyperresponsiveness in familial hypercholesterolemia arises from an alteration in the coupling mechanism between thrombin binding and response such that platelets from patients with familial hypercholesterolemia are able to respond with lower receptor occupancy than is the case with normal platelets. Thrombin binding and aggregation were within normal ranges for platelets from abetalipoproteinemia patients (N = 4) and type V hyperlipoproteinemia (N = 2), although in the latter case the response appeared to be less at very low thrombin concentrations (less than 30 mU/mL). Thrombin binding was elevated in Tangier disease (N = 3) but with lower responsiveness at lower thrombin concentrations. Thrombin binding was also elevated in LCAT deficiency (N = 2), and one patient showed increased and another showed decreased aggregation responses. In general, increased plasma cholesterol levels resulted in increased stimulus-response coupling (type II), whereas increased triglyceride levels resulted in decreased coupling (type V, Tangier), and there was no apparent alteration in the coupling mechanism with overall reduction in plasma lipid levels as in abetalipoproteinemia.

Thrombin binding and response in platelets from patients with dyslipoproteinemias: increased stimulus-response coupling in type II hyperlipoproteinemia

Platelets were obtained from patients with various hyperlipidemias [type II, type V, lecithin-cholesterol acyltransferase (LCAT) deficiency] and hypolipidemias (abetalipoproteinemia, Tangier disease) to ascertain relationships among plasma lipids, platelet lipids, thrombin binding and thrombin-induced platelet aggregation, and to compare these data with those previously obtained on stimulus-response coupling in platelets following in vitro modification of membrane microviscosity. Washed platelets were studied for their ability to bind 125I-thrombin in the range of 10(-10) to 10(-6) mol/L (10 mU/mL to 100 U/mL) and to aggregate with thrombin at concentrations less than 10(-9) mol/L (100 mU/mL). The values for binding and aggregation in eight patients from six kindred with familial hypercholesterolemia, taken as a group, fell in the low normal range. If divided into two groups, patients with overt cardiovascular disease bound normal amounts of thrombin but were more responsive to it, whereas patients without overt cardiovascular disease bound lower amounts of thrombin but gave an aggregation response in the normal range. These results suggest that platelet hyperresponsiveness in familial hypercholesterolemia arises from an alteration in the coupling mechanism between thrombin binding and response such that platelets from patients with familial hypercholesterolemia are able to respond with lower receptor occupancy than is the case with normal platelets. Thrombin binding and aggregation were within normal ranges for platelets from abetalipoproteinemia patients (N = 4) and type V hyperlipoproteinemia (N = 2), although in the latter case the response appeared to be less at very low thrombin concentrations (less than 30 mU/mL). Thrombin binding was elevated in Tangier disease (N = 3) but with lower responsiveness at lower thrombin concentrations. Thrombin binding was also elevated in LCAT deficiency (N = 2), and one patient showed increased and another showed decreased aggregation responses. In general, increased plasma cholesterol levels resulted in increased stimulus-response coupling (type II), whereas increased triglyceride levels resulted in decreased coupling (type V, Tangier), and there was no apparent alteration in the coupling mechanism with overall reduction in plasma lipid levels as in abetalipoproteinemia.

Aggregation and thromboxane B 2 formation in platelets and vascular prostacyclin production from genetically obese rats

Obesity, diabetes, hyperlipidaemia and age are conditions predisposing to atheroscleorosis and arterial occlusion. Recently it has been claimed that increased synthesis of thromboxane A 2 by platelets and decreased synthesis of prostacyclin (PGI 2) by blood vessels play an important role. The “Zucker” rat, a genetically obese animal with hyperlipidaemia, hyperinsulinaemia and normoglycaemia was used to study platelet aggregation, thromboxane (TXB 2) production and aortic PGI 2 synthesis. Two age groups (6–8 months and 14–16 months old) and their homozygote lean controls were used. In the obese rats no increased aggregation was found with ADP, arachidonic acid and collagen. On the contrary platelets from young fatty rats were less sensitive to ADP than platelets from lean young animals. An increase in platelet sensitivity to aggregating agents with age was observed, especially in the obese rats. TXB 2 measured in platelet rich plasma after exposure to ADP, arachidonic acid, arachidonic acid plus ADP and collagen was similar in the fatty and lean animals. Production of PGI 2 from incubated aortic rings was lowest in young lean animals. No differences existed between the other groups of rats studied. Insulin added to aortic rings had no influence on PGI 2 production. It is concluded that age rather than obesity, hyperlipidaemia or hyperinsulinaemia may cause platelet hyperresponsiveness to aggregating agents. Thromboxane and plateletaggregation do not closely correlate. PGI 2 production is not reduced by metabolic alterations, thought to predispose to atherosclerosis.

Platelet abnormalities in ischemic heart disease

Forty-seven patients with well documented ischemic heart disease were studied with tests designed to detect “hypercoagulability”: platelet count, platelet aggregation response to dilutions of adenosine diphosphate (ADP) and collagen, determination of fibrinolytic activity and thromboelastography. No abnormalities were found in the thromboelastograms, fibrinolytic activity, platelet count or response of platelets to ADP. Most patients demonstrated abnormal platelet reactivity to collagen soon after myocardial infarction. Five patients (but none of the normal subjects or patients with nonischemic heart disease) had a persistent marked abnormality, with platelet hyperresponsiveness to collagen still present 10 to 16 months after infarction. These five patients differed from others with myocardial infarction by being younger, having a paucity of the recognized risk factors and having either normal coronary arteriograms or single obstructive arterial lesions. Each had significantly decreased 51chromium platelet survival time many months after infarction, during an asymptomatic period. One of eight patients with chest pain of the anginal type and normal coronary arteriograms showed similar platelet hyperaggregability. We suggest that some patients with ischemic heart disease, perhaps as many as 10 percent, have chronic abnormal platelet function, best detected by the platelet aggregation response to dilutions of collagen and by platelet survival studies. This may be a primary abnormality in patients with ischemic heart disease whose arteriograms show no advanced coronary atherosclerotic obstructive lesions. Forty-seven patients with well documented ischemic heart disease were studied with tests designed to detect “hypercoagulability”: platelet count, platelet aggregation response to dilutions of adenosine diphosphate (ADP) and collagen, determination of fibrinolytic activity and thromboelastography. No abnormalities were found in the thromboelastograms, fibrinolytic activity, platelet count or response of platelets to ADP. Most patients demonstrated abnormal platelet reactivity to collagen soon after myocardial infarction. Five patients (but none of the normal subjects or patients with nonischemic heart disease) had a persistent marked abnormality, with platelet hyperresponsiveness to collagen still present 10 to 16 months after infarction. These five patients differed from others with myocardial infarction by being younger, having a paucity of the recognized risk factors and having either normal coronary arteriograms or single obstructive arterial lesions. Each had significantly decreased 51chromium platelet survival time many months after infarction, during an asymptomatic period. One of eight patients with chest pain of the anginal type and normal coronary arteriograms showed similar platelet hyperaggregability. We suggest that some patients with ischemic heart disease, perhaps as many as 10 percent, have chronic abnormal platelet function, best detected by the platelet aggregation response to dilutions of collagen and by platelet survival studies. This may be a primary abnormality in patients with ischemic heart disease whose arteriograms show no advanced coronary atherosclerotic obstructive lesions.