BackgroundMore 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.ObjectiveThis study was designed to determine if blood biomarkers could provide objective criteria to assist diagnosis of GWI.DesignA 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.ResultsLymphocyte, 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%.SignificanceThe 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.

Human platelet thromboxane A2/prostaglandin H2 (TXA2/ PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G pro-, teins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2-platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, 1251IBOP and 13H1U46619, and antagonist, 13H1SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2-platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2-platelets. Antisera to Gq class a subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTPyS yielded significantly less PA and IP3 in TXA2-platelets. Immunotransfer blotting revealed comparable quantities of Gq class a-subunits in all three platelet types. Thus, TXA2-dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a

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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 A 2 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 lM) 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 lM) 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 lM blebbistatin.

SummaryMost mongrel dogs have platelets that form thromboxane A2 (TXA2) from exogenous arachidonate, but they fail to aggregate or secrete in response to it. In contrast to these TXA2 insensitive (TXA2-) platelets, some dogs have TXA2 sensitive (TXA2+) platelets that aggregate and secrete when stirred with arachidonate. To evaluate the possible genetic basis for this difference, we carried out seven matings of mongrel dogs that yielded 48 viable offspring. Four matings of dogs with TXA2- platelets (presumed genotype TT) including 2 back-crosses, produced 32 pups with TXA2- (TT) platelets and 0 pups with TXA2+ platelets. A cross between a male with TXA2+ platelets (presumed genotype tt) and a female with TXA2+ (tt) platelets yielded 9 offspring with TXA2+ (tt) platelets and 0 with TXA2- platelets. Crossing a male presumed homozygous (TT) for TXA2- platelets with a female with TXA2+ (tt) platelets produced 2 pups with TXA2- (Tt) platelets and 0 pups with TXA2+ (tt) platelets. The same female with TXA2+ platelets crossed with a male presumed to be heterozygous (Tt) for TXA2- platelets yielded 2 pups with TXA2+ (tt) platelets and 3 pups with TXA2- (Tt) platelets. Segregation analysis of these data supports the hypothesis that the ability of dog platelets to aggregate and secrete in response to TXA2 is inherited as an autosomal recessive trait.

G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is not responsible for defective activation of PI-PLC in thromboxane-insensitive dog platelets. Despite the high degree of similarity that exists between G alpha q and G alpha 11, significantly greater species-specific variation in nucleotide sequence is present in G alpha 11 than in G alpha q. Cellular specificity and species specificity are important characteristics of these Gq family G-proteins.

A previous study of Gulf War veteran's 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 μmol/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 μmol/l U46619 was significantly greater in patients whose CRP was at least 2 μg/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.

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.