Linda A. Leis

Arachidonate-induced platelet aggregation in the dog

Abstract The platelet rich plasma (PRP) from most dogs aggregates in response to ADP, collagen and thrombin. Dog PRP generally does not aggregate in response to epinephrine, and previous studies found that dog PRP uniformly failed to aggregate when exposed to arachidonic acid prepared in an ethanol-sodium carbonate medium. Our studies demonstrate that 30% of randomly selected mongrel dogs have PRP which aggregates when exposed to sodium arachidonate dissolved in modified Tyrodes buffer, PRP from these dogs also aggregates with lower concentrations of ADP and collagen than PRP which is unresponsive to arachidonate. Pre-incubation of PRP with epinephrine uniformly transforms PRP which does not aggregate on exposure to arachidonate alone into arachidonate-aggregating PRP. Dog PRP which aggregates with arachidonate release 14 C-serotonin, while non-aggregating PRP does not. However, arachidonate stimulates malondialdehyde production in both aggregating and non-aggregating PRP. The results of this study indicate that dogs are heterogeneous in regard to their aggregation response to arachidonate. The mechanism of this heterogeneity is unknown: however, since prostaglandin metabolism is intact and the platelets of some dogs respond to arachidonate alone, it appears to be the result of variable sensitivity to endoperoxides and thromboxane A 2 .

The critical role of myosin IIA in platelet internal contraction.

Summary.  Background: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined.Objective: To define the contribution of myosin IIA to platelet internal contraction.Methods: Aspirin‐treated platelets suspended in calcium‐free buffer were activated with a low concentration (25 nm) of the thromboxane A2 analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently.Results and Conclusions: Low‐concentration blebbistatin (10 μm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25–100 μm) produced concentration‐dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 μm blebbistatin.

Tissue factor activity of blood mononuclear cells is increased after total knee arthroplasty

Tissue factor (TF) is present in small quantities in normal blood and is reported to be elevated in arterial and venous thrombosis. Patients undergoing total knee arthoplasty (TKA) are at high risk of post-operative venous thromboembolism (VTE). To evaluate the possible contribution of elevated blood TF to VTE risk, we performed serial studies of peripheral blood mononuclear cell (PBMC) functional TF procoagulant activity (PCA) in 19 patients after TKA. PBMC and platelet TF PCA were measured by a functional, clot-based assay following decryption with a calcium ionophore. Plasma TF antigen levels were measured by ELISA. All subjects received chemoprophylaxis and none had VTE. After TKA total TF PCA of PBMC was elevated in 19 of 19 subjects. The peak increase above preoperative levels was 1.1-13.6 fold (>two-fold in 58% and >three-fold in 42%). Median TF PCA of PBMC was not elevated following tourniquet removal, but it was significantly elevated on postoperative days 1 and 2. Thereafter, it decreased to near preoperative values at day 6. Neither platelet TF PCA nor plasma TF antigen levels increased significantly. Since the PBMC count did not rise, the increase in TF PCA was attributable to cell synthesis. The increase in blood TF PCA preceded the median time of diagnosis of venous thromboembolism after TKA established previously. These observations indicate a) TKA stimulates synthesis of encrypted PBMC TF PCA which is likely to contribute to the pathophysiology of VTE; b) TF antigen is not a reliable indicator of TF PCA.

Blood Biomarkers of Chronic Inflammation in Gulf War Illness.

Background More than twenty years following the end of the 1990–1991 Gulf War it is estimated that approximately 300,000 veterans of this conflict suffer from an unexplained chronic, multi-system disorder known as Gulf War Illness (GWI). The etiology of GWI may be exposure to chemical toxins, but it remains only partially defined, and its case definition is based only on symptoms. Objective criteria for the diagnosis of GWI are urgently needed for diagnosis and therapeutic research. Objective This study was designed to determine if blood biomarkers could provide objective criteria to assist diagnosis of GWI. Design A surveillance study of 85 Gulf War Veteran volunteers identified from the Department of Veterans Affairs Minnesota Gulf War registry was performed. All subjects were deployed to the Gulf War. Fifty seven subjects had GWI defined by CDC criteria, and 28 did not have symptomatic criteria for a diagnosis of GWI. Statistical analyses were performed on peripheral blood counts and assays of 61 plasma proteins using the Mann-Whitney rank sum test to compare biomarker distributions and stepwise logistic regression to formulate a diagnostic model. Results Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects. A diagnostic model of three biomarkers—lymphocytes, monocytes, and C reactive protein—had a predicted probability of 90% (CI 76–90%) for diagnosing GWI when the probability of having GWI was above 70%. Significance The results of the current study indicate that inflammation is a component of the pathobiology of GWI. Analysis of the data resulted in a model utilizing three readily measurable biomarkers that appears to significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials.

Elevated platelet count, C-reactive protein and thromboxane analog-induced platelet aggregation in patients with Gulf War veterans' illnesses: evidence of a chronic inflammatory state?

A previous study of Gulf War veterans illnesses (GWVI) observed evidence of platelet activation in a majority of patients with GWVI. To further characterize platelet function, we studied 43 patients (40 men) with GWVI (GWVI+) and 21 veterans who served concurrently in the Gulf War but who lacked criteria for GWVI (GWVI−). All participants were free of infection and known inflammatory diseases. Studies performed included platelet count, immature platelet fraction (IPF), plasma thrombopoietin (TPO), C-reactive protein (CRP), platelet aggregation and ATP secretion in response to six agonists, and spontaneous aggregation. Platelet counts and CRP were significantly elevated in GWVI+ compared to GWVI− patients without elevation in IPF or TPO. Platelet aggregation did not differ between GWVI+ and GWVI− patients except for spontaneous aggregation that was significantly greater in GWVI+ patients. Platelet ATP secretion was similar in the two groups, except the response to 50 &mgr;mol/l thrombin receptor agonist peptide 6 (TRAP 6) was significantly greater in GWVI+ patients. When platelet aggregation was analyzed in relation to CRP, the response to 0.5 &mgr;mol/l U46619 was significantly greater in patients whose CRP was at least 2 &mgr;g/ml. Therefore, GWVI+ patients had elevated platelet counts, spontaneous aggregation, TRAP 6-induced secretion, and CRP, but no impairment of platelet function. The increased platelet counts and U46619-induced aggregation appear to be consequences of an underlying inflammatory state in GWVI.

Adenosine potentiates the inhibitory effects of calcium channel antagonists on human platelet aggregation induced by thromboxane A2 or U46619

Calcium channel antagonists inhibit platelet function in vitro and ex vivo, but the mechanism responsible has not been clearly defined. The concentrations of these agents required to inhibit platelet aggregation in vitro are several fold higher than those attained in vivo. Adenosine, a known inhibitor of platelet function, is produced in large quantities in ischemic myocardium. In order to test the hypothesis that adenosine may potentiate the platelet-inhibitory effects of calcium channel antagonists, we studied the effect of adenosine plus nifedipine, verapamil or diltiazem on human platelet aggregation induced by thromboxane A2 or the stable endoperoxide/thromboxane A2 mimic, U46619 +/- epinephrine. Adenosine, in concentrations achieved in the plasma during myocardial ischemia (0.01-0.1 microM), enhanced the inhibitory effects of nifedipine, verapamil and diltiazem on platelet aggregation 5-100 fold. The same concentrations of adenosine alone did not inhibit platelet aggregation. In the presence of non-inhibitory concentrations of adenosine, nifedipine, in concentrations approaching those attained in vivo following standard therapeutic doses (as low as 0.29 microM), significantly inhibited thromboxane A2-induced platelet aggregation. Therefore, adenosine potentiates the in vitro inhibitory effects of calcium channel antagonists on platelet aggregation induced by thromboxane A2 or thromboxane A2 plus epinephrine. These results suggest that adenosine production by ischemic myocardium may augment the inhibitory effect of calcium channel antagonists on platelets.