Hemocytometric characteristics of COVID-19 patients with and without cytokine storm syndrome on the sysmex XN-10 hematology analyzer.

OBJECTIVES: COVID-19 is an ongoing global pandemic. There is an urgent need for identification and understanding of clinical and laboratory parameters related to progression towards a severe and fatal form of this illness, often preceded by a so-called cytokine-storm syndrome (CSS). Therefore, we explored the hemocytometric characteristics of COVID-19 patients in relation to the deteriorating clinical condition CSS, using the Sysmex XN-10 hematology analyzer. METHODS: From March 1st till May 16th, 2020, all patients admitted to our hospital with respiratory complaints and suspected for COVID-19 were included (n=1,140 of whom n=533 COVID-19 positive). The hemocytometric parameters of immunocompetent cells in peripheral blood (neutrophils [NE], lymphocytes [LY] and monocytes [MO]) obtained upon admission to the emergency department (ED) of COVID-19 positive patients were compared with those of the COVID-19 negative ones. Moreover, patients with CSS (n=169) were compared with COVID-19 positive patients without CSS, as well as with COVID-19 negative ones. RESULTS: In addition to a significant reduction in leukocytes, thrombocytes and absolute neutrophils, it appeared that lymphocytes-forward scatter (LY-FSC), and reactive lymphocytes (RE-LYMPHO)/leukocytes were higher in COVID-19-positive than negative patients. At the moment of presentation, COVID-19 positive patients with CSS had different neutrophils-side fluorescence (NE-SFL), neutrophils-forward scatter (NE-FSC), LY-FSC, RE-LYMPHO/lymphocytes, antibody-synthesizing (AS)-LYMPHOs, high fluorescence lymphocytes (HFLC), MO-SSC, MO-SFL, and Reactive (RE)-MONOs. Finally, absolute eosinophils, basophils, lymphocytes, monocytes and MO-FSC were lower in patients with CSS. CONCLUSIONS: Hemocytometric parameters indicative of changes in immunocompetent peripheral blood cells and measured at admission to the ED were associated with COVID-19 with and without CSS.

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

RATIONALE: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbß3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. OBJECTIVE: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. METHODS AND RESULTS: JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbß3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbß3 signaling in vitro. CONCLUSIONS: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets.

BACKGROUND: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. METHODS AND RESULTS: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. CONCLUSIONS: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbß3 and transglutaminase factor XIII.

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)ß3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)ß3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)ß3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)ß3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)ß3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)ß3.

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase ß, and Not Via CD40 and IκB Kinase α.

OBJECTIVE: To investigate the roles and signaling pathways of CD40L and CD40 in platelet-platelet interactions and thrombus formation under conditions relevant for atherothrombosis. APPROACH AND RESULTS: Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg(-/-)Apoe(-/-)) showed diminished αIIbß3 activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40(-/-)Apoe(-/-)) were not decreased. Using blood from Cd40lg(-/-)Apoe(-/-) and Cd40(-/-)Apoe(-/-) mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe(-/-) blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI-induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb(R/R) platelets or by inhibiting phosphatidylinositol 3-kinase ß (PI3Kß). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kß. Finally, platelets from Chuk1(A/A)Apoe(-/-) mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation. CONCLUSIONS: Under atherogenic conditions, CD40L enhances collagen-induced platelet-platelet interactions by supporting integrin αIIbß3 activation, secretion and thrombus growth via PI3Kß, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation.

Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation.

Scott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis- or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets. High, but not moderate, PS exposure in platelets was suppressed by chelation of intracellular Ca(2+), whereas caspase inhibition completely abolished ABT737-induced PS exposure in both Scott and control platelets. On the other hand, high PS exposure induced by the Ca(2+)-mobilizing agonists convulxin/thrombin fully relied on mitochondrial depolarization and was virtually absent in Scott platelets. Finally, PS exposure induced by collagen/thrombin was partly affected in Scott platelets, and the residual PS positive fraction was insensitive to inhibition of caspases or mitochondrial depolarization. In conclusion, TMEM16F is not required for, but enhances, caspase-dependent PS exposure; convulxin-/thrombin-induced PS exposure is entirely dependent on TMEM16F, whereas collagen/thrombin-induced PS exposure results from 2 distinct pathways, one of which involves mitochondrial depolarization and is mediated by TMEM16F.

Factor XII regulates the pathological process of thrombus formation on ruptured plaques.

OBJECTIVE: Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo. APPROACH AND RESULTS: Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas. CONCLUSIONS: The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.

Dual mechanism of integrin αIIbß3 closure in procoagulant platelets.

BACKGROUND: Inactivation of integrin αIIbß3 reverses platelet aggregate formation upon coagulation. RESULTS AND CONCLUSION: Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbß3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. SIGNIFICANCE: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbß3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbß3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the ß3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbß3 inactivation. Integrin αIIbß3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbß3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbß3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbß3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.

The effects of arterial flow on platelet activation, thrombus growth, and stabilization.

Injury of an arterial vessel wall acutely triggers a multifaceted process of thrombus formation, which is dictated by the high-shear flow conditions in the artery. In this overview, we describe how the classical concept of arterial thrombus formation and vascular occlusion, driven by platelet activation and fibrin formation, can be extended and fine-tuned. This has become possible because of recent insight into the mechanisms of: (i) platelet-vessel wall and platelet-platelet communication, (ii) autocrine platelet activation, and (iii) platelet-coagulation interactions, in relation to blood flow dynamics. We list over 40 studies with genetically modified mice showing a role of platelet and plasma proteins in the control of thrombus stability after vascular injury. These include multiple platelet adhesive receptors and other junctional molecules, components of the ADP receptor signalling cascade to integrin activation, proteins controlling platelet shape, and autocrine activation processes, as well as multiple plasma proteins binding to platelets and proteins of the intrinsic coagulation cascade. Regulatory roles herein of the endothelium and other blood cells are recapitulated as well. Patient studies support the contribution of platelet- and coagulation activation in the regulation of thrombus stability. Analysis of the factors determining flow-dependent thrombus stabilization and embolus formation in mice will help to understand the regulation of this process in human arterial disease.

Calcitonin gene-related peptide terminates long-lasting vasopressor responses to endothelin 1 in vivo.

Slow dissociation of endothelin 1 from its endothelin A receptors is responsible for the long-lasting vasoconstrictor effects of the peptide. We showed recently that calcitonin gene-related peptide selectively terminates long-lasting contractile responses to endothelin 1 in isolated rat mesenteric arteries. Here we assessed whether the antiendothelinergic effect of calcitonin gene-related peptide is vascular bed specific and may terminate long-lasting pressor responses to exogenous and locally produced endothelin 1 in vivo. Regional heterogeneity of the calcitonin gene-related peptide/endothelin A receptor cross-talk was explored in arteries isolated from various rat organs. Endothelin A receptor-mediated arterial contractions were terminated by calcitonin gene-related peptide in rat mesenteric, renal, and spermatic arteries but not in basilar, coronary, epigastric, gastric, splenic, and saphenous arteries. Endothelin A receptor antagonism only ended endothelin 1-induced contractions in spermatic arteries. In anesthetized rats, instrumented with Doppler flow probes to record regional blood flows, long-lasting pressor and vasoconstrictor responses to an intravenous bolus injection of endothelin 1 or big endothelin 1 were transiently reduced by sodium nitroprusside (NO donor) but terminated by intravenously administered calcitonin gene-related peptide. In conscious rats, calcitonin gene-related peptide but not sodium nitroprusside terminated prolonged (>60-minute) pressor responses to endothelin 1 but not those to intravenous infusion of phenylephrine. In conclusion, pressor responses to circulating and locally produced endothelin 1 that are resistant to endothelin receptor antagonism and NO can be terminated by a regionally selective effect of calcitonin gene-related peptide. Calcitonin gene related peptide receptor agonism may represent a novel strategy to treat endothelin 1-associated cardiovascular pathologies.