The role of bone marrow-derived cells in venous thromboembolism.

Venous thrombosis is a life-threatening condition with high morbidity and mortality. Abnormal functioning of different cells in the blood is an integral part of its pathogenesis. In this review, we describe the contribution of bone marrow-derived cells to the development of this debilitating disease. We present both epidemiological and clinical data demonstrating involvement of various cell types in venous thrombosis, and discuss potential mechanisms underlying these effects. Modern concepts including recently discovered new paradigms in thrombosis, such as neutrophil extracellular traps, mast cells, and polyphosphate, are summarized.

Pulmonary Artery Thrombosis: A Diagnosis That Strives for Its Independence.

According to a widespread theory, thrombotic masses are not formed in the pulmonary artery (PA) but result from migration of blood clots from the venous system. This concept has prevailed in clinical practice for more than a century. However, a new technologic era has brought forth more diagnostic possibilities, and it has been shown that thrombotic masses in the PA could, in many cases, be found without any obvious source of emboli. Chronic obstructive pulmonary disease, asthma, sickle cell anemia, emergency and elective surgery, viral pneumonia, and other conditions could be complicated by PA thrombosis development without concomitant deep vein thrombosis (DVT). Different pathologies have different causes for local PA thrombotic process. As evidenced by experimental results and clinical observations, endothelial and platelet activation are the crucial mechanisms of this process. Endothelial dysfunction can impair antithrombotic function of the arterial wall through downregulation of endothelial nitric oxide synthase (eNOS) or via stimulation of adhesion receptor expression. Hypoxia, proinflammatory cytokines, or genetic mutations may underlie the procoagulant phenotype of the PA endothelium. Both endotheliocytes and platelets could be activated by protease mediated receptor (PAR)- and receptors for advanced glycation end (RAGE)-dependent mechanisms. Hypoxia, in particular induced by high altitudes, could play a role in thrombotic complications as a trigger of platelet activity. In this review, we discuss potential mechanisms of PA thrombosis in situ.

Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

Podoplanin regulates the migration of mesenchymal stromal cells and their interaction with platelets.

Mesenchymal stromal cells (MSCs) upregulate podoplanin at sites of infection, chronic inflammation and cancer. Here, we investigated the functional consequences of podoplanin expression on the migratory potential of MSCs and their interactions with circulating platelets. Expression of podoplanin significantly enhanced the migration of MSCs compared to MSCs lacking podoplanin. Rac-1 inhibition altered the membrane localisation of podoplanin and in turn significantly reduced MSC migration. Blocking Rac-1 activity had no effect on the migration of MSCs lacking podoplanin, indicating that it was responsible for regulation of migration through podoplanin. When podoplanin-expressing MSCs were seeded on the basal surface of a porous filter, they were able to capture platelets perfused over the uncoated apical surface and induce platelet aggregation. Similar microthrombi were observed when endothelial cells (ECs) were co-cultured on the apical surface. Confocal imaging shows podoplanin-expressing MSCs extending processes into the EC layer, and these processes could interact with circulating platelets. In both models, platelet aggregation induced by podoplanin-expressing MSCs was inhibited by treatment with recombinant soluble C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b). Thus, podoplanin may enhance the migratory capacity of tissue-resident MSCs and enable novel interactions with cells expressing CLEC-2.

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

Mast Cells Granular Contents Are Crucial for Deep Vein Thrombosis in Mice.

RATIONALE: Deep vein thrombosis (DVT) and its complication pulmonary embolism have high morbidity reducing quality of life and leading to death. Cellular mechanisms of DVT initiation remain poorly understood. OBJECTIVE: We sought to determine the role of mast cells (MCs) in DVT initiation and validate MCs as a potential target for DVT prevention. METHODS AND RESULTS: In a mouse model, DVT was induced by partial ligation (stenosis) of the inferior vena cava. We demonstrated that 2 strains of mice deficient for MCs were completely protected from DVT. Adoptive transfer of in vitro differentiated MCs restored thrombosis. MCs were present in the venous wall, and the number of granule-containing MCs decreased with thrombosis. Pharmacological depletion of MCs granules or prevention of MC degranulation also reduced DVT. Basal plasma levels of von Willebrand factor and recruitment of platelets to the inferior vena cava wall after DVT induction were reduced in MC-deficient mice. Stenosis application increased plasma levels of soluble P-selectin in wild-type but not in MC-deficient mice. MC releasate elevated ICAM-1 (intercellular adhesion molecule-1) expression on HUVEC (human umbilical vein endothelial cells) in vitro. Topical application of compound 48/80, an MC secretagogue, or histamine, a Weibel-Palade body secretagogue from MCs, potentiated DVT in wild-type mice, and histamine restored thrombosis in MC-deficient animals. CONCLUSIONS: MCs exacerbate DVT likely through endothelial activation and Weibel-Palade body release, which is, at least in part, mediated by histamine. Because MCs do not directly contribute to normal hemostasis, they can be considered potential targets for prevention of DVT in humans.

Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice.

Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) the proteolytically active intermediate, meizothrombin (fIIa(MZ)). FIIa(MZ) has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIa(MZ), mutations were introduced into the endogenous fII gene, resulting in expression of prothrombin carrying 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa(MZ) Homozygous fII(MZ) mice are viable, express fII levels comparable with fII(WT) mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIa(MZ) enzyme activity, platelet aggregation by fII(MZ) is similar to fII(WT) Consistent with prior analyses of human fIIa(MZ), significant prolongation of clotting times was observed for fII(MZ) plasma. Adult fII(MZ) animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fII(MZ) mice had 2 significant phenotypic advantages over fII(WT) animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIa(MZ) in vivo.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

Neutrophil extracellular trap (NET) impact on deep vein thrombosis.

Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment. Inflammatory conditions such as infection, cancer, and autoimmune diseases are risk factors for DVT. We and others have recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellular traps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant in thrombi in animal models of DVT. It appears that, in addition to fibrin and von Willebrand factor, NETs represent a third thrombus scaffold. Here, we review how NETs stimulate thrombosis and discuss known and potential interactions of NETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis. We propose that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT.

p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

AIMS: Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. METHODS AND RESULTS: Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. CONCLUSION: Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

Platelet-derived microparticles promote invasiveness of prostate cancer cells via upregulation of MMP-2 production.

Prostate cancer commonly affects men in the Western world. A major factor of the life-threatening course of this disease is the high rate of metastasis, predominantly to bones. Circulating tumor cells encounter platelets and may activate them, resulting in a production of microparticles (MPs). MPs are small platelet fragments expressing membrane receptors as well as cytoplasmic constituents. Here, we report that prostate cancer cells, Clone-1 (Cl-1), preincubated with platelet-derived MPs (PMPs), demonstrate increased invasion through a gelatin-coated (a denatured form of collagen) membrane of the Boyden chamber system. This effect was accompanied by an increased secretion of metalloproteinase-2 (MMP-2) as demonstrated by a gelatin zymography. Application of MMP-2/9 inhibitor reversed the PMP-induced tumor cell invasion. PMPs were shown to adhere to Cl-1 cells, but direct contact between them may not be mandatory for MMP secretion because PMP lysate induced MMP-2 production by Cl-1 cells to the same extent as did intact PMPs. PMP-induced MMP-2 secretion was inhibited by neutralization of either PKC or total intracellular tyrosine phosphorylation, but was not affected by blocking major intraplatelet cytokines. Actinomycin D (a transcription inhibitor) did not modify this effect, whereas cycloheximide (an inhibitor of protein translation) abolished the MMP-2 release. MMP-2 secretion was accompanied by a rapid and transient increase in MMP-2 mRNA level after a 2-hr coincubation of prostate cancer cells with PMPs. Thus, PMPs promote tumor invasiveness, at least in part by stimulation of MMP-2 production.

Effects of estradiol and raloxifene on arterial thrombosis in ovariectomized mice.

OBJECTIVE: The effects of estrogen and selective estrogen receptor modulators (eg, raloxifene) on arterial thrombosis are not well defined. This study assessed the manner and mechanism by which estrogen and raloxifene affect homeostatic pathways in ovariectomized mice after acute arterial injury. DESIGN: Female mice (3 weeks old) underwent ovariectomy or sham operation. Five days after surgery, mice were assigned to treatment with estradiol (5.3 nmol/kg), raloxifene (2.7 micromol/kg), or placebo (n = 10-12/group). The biological effects of both treatments were assessed by measurements of bone mass and the degree of uterine atrophy. After 4 months of therapy, carotid artery thrombosis was induced by photochemical injury, and the time to vascular occlusion was measured. RESULTS: Both treatments increased bone mineral density (4.1%-7.85%). Reversal of macroscopic uterine atrophy was observed only in estrogen-treated mice. Ovariectomized mice had a shorter time to occlusion compared with sham-operated mice (70.8 +/- 7.4 vs 103 +/- 11.3 min), suggesting accelerated thrombosis. Both estradiol and raloxifene significantly inhibited intra-arterial thrombosis in ovariectomized mice, prolonging the time to occlusion to 136.33 +/- 13.5 and 141.43 +/- 9.26 min, respectively. Cyclooxygenase-2 levels in the lung tissue were significantly increased by both raloxifene and estradiol with endothelial nitric oxide synthase expression being unaltered. Platelet adhesion (measured by surface coverage under a shear rate of 1,800 s for 2 min) was significantly reduced in ovariectomized animals, being 4.63% +/- 1.47%, 5.78% +/- 1.58%, and 10.04% +/- 1.33% for raloxifene, estradiol, and placebo, respectively. CONCLUSIONS: Ovariectomy amplifies thrombosis. We found that 4 months of treatment with both estradiol and raloxifene attenuates intravascular thrombosis. The antithrombotic effect was accompanied by increased expression of cyclooxygenase-2 and suppression of platelet surface adhesion.

Functional CXCR4-expressing microparticles and SDF-1 correlate with circulating acute myelogenous leukemia cells.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4(+) microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4(+) microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4(+) microparticles in AML patients were CD45(+), whereas in normal individuals, they were mostly CD41(+). Importantly, we found a strong correlation between the levels of CXCR4(+) microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH(2)-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/beta2m(null) mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4(+) microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable.

Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing.

We develop an optical whole-body imaging technique for monitoring normal and leukemic hematopoietic cell homing in vivo. A recently developed near-infrared (NIR) lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) is used to safely and directly label the membranes of human leukemic Pre-B ALL G2 cell lines as well as primary murine lymphocytes and erythrocytes. DiR has absorption and fluorescence maxima at 750 and 782 nm, respectively, which corresponds to low light absorption and autofluorescence in living tissues. This allows us to obtain a significant signal with very low background level. A charge-coupled device (CCD)-based imager is used for noninvasive whole-body imaging of DiR-labeled cell homing in intact animals. This powerful technique can potentially visualize any cell type without use of specific antibodies conjugated with NIR fluorescent tag or loading cells with transporter-delivered NIR fluorophores. Thus, in vivo imaging based on NIR lipophilic carbocyanine dyes in combination with advanced optical techniques may serve as a powerful alternative or complementation to other small animal imaging methods.

C-reactive protein promotes platelet adhesion to endothelial cells: a potential pathway in atherothrombosis.

C-reactive protein (CRP) is a strong predictor for acute cardiovascular events. Several endothelial prothrombotic effects of CRP have been recently reported. This study examined the effect of CRP on bovine aortic endothelial cell (EC) activation and capacity to recruit human platelets under flow conditions using the cone and plate(let) analyser method. Human recombinant CRP promoted platelet adhesion in a dose- and time-dependent manner, with a maximal effect at 20 microg/ml (increase of 174% over baseline, P < 0.01). Similar effects were observed following incubation of EC with sera of transgenic mice that express human CRP (10 microg/ml). Anti-intercellular adhesion molecule-1 neutralising monoclonal antibody and nitric oxide donor, sodium nitroprusside, blocked the effect of CRP, reducing adhesion from 202% to 128% (P < 0.05) and 114% (P = 0.02) respectively. The pro-adhesive effect of CRP was abolished by calphostin C (a protein kinase C inhibitor), whereas the extracellular signal-regulated kinase antagonist, PD98059, did not have any effect. CRP promoted P-selectin expression on the EC surface and blockade of P-selectin reversed CRP-induced platelet adhesion. In conclusion, CRP promoted platelet adhesion to EC. Our results emphasise the possible role of CRP in linking inflammation and thrombosis and provide a potential mechanism for the high incidence of vascular events associated with high CRP levels.

A new point-of-care method for monitoring anti-platelet therapy: application of the cone and plate(let) analyzer.

Recent studies suggest that anti-platelet agents are not equally effective in all individuals. We have developed a new method to evaluate the effect of anti-platelet drugs using the cone and plate(let) analyzer (CPA) test. The method is based on the ability of activators to reduce platelet adhesion under flow conditions. Treatment of a blood sample with arachidonic acid (AA) or ADP in vitro significantly decreased platelet deposition to a surface coverage (SC) of 2.1+/-0.4 and 1.3+/-0.6%, respectively, compared with the basic SC of 12.3+/-6.8%. The effect of AA was prevented by aspirin (SC 8.1+/-3.8%) and that of ADP was reduced by 2-methylthio-AMP, a P2Y12 ADP receptor inhibitor (SC 4.8+/-2.0%). Pre-incubation with AA of whole blood samples from untreated healthy volunteers resulted in a marked decline of SC (from SC 9.8+/-2.2 to 0.6+/-0.3%). In contrast, in volunteers treated with 100, 300, and 500 mg aspirin per day, AA (but not ADP) decreased SC only to 3.5+/-1.3, 4.4+/-1.7, and 4.1+/-2.0%, respectively (P<0.001 versus SC with AA before treatment). A good correlation was observed between the modified CPA and aggregometry (R2=0.55). In conclusion, the modified CPA test is a useful tool to evaluate the efficacy of anti-platelet therapy.