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Antiplatelet drugs, such as aspirin, P2Y 12 antagonists, and glycoprotein (GP) IIb/IIIa inhibitors, have proved to be successful in reducing the morbidity and mortality associated with arterial thrombosis. These agents are, therefore, the cornerstone of therapy for patients with acute coronary syndromes. However, these drugs all carry an inherent risk of bleeding, which is associated with adverse cardiovascular outcomes and mortality. Thus, the potential benefits of more potent, conventional antiplatelet drugs are likely be offset by the increased risk of bleeding. Data from experiments in vivo have highlighted potentially important differences between haemostasis and thrombosis, raising the prospect of developing new antiplatelet drugs that are not associated with bleeding. Indeed, in preclinical studies, several novel antiplatelet therapies that seem to inhibit thrombosis while maintaining haemostasis have been identified. These agents include inhibitors of phosphatidylinositol 3-kinase-β (PI3Kβ), protein disulfide-isomerase, activated GPIIb/IIIa, GPIIb/IIIa outside-in signalling, protease-activated receptors, and platelet GPVI-mediated adhesion pathways. In this Review, we discuss how a therapeutic ceiling has been reached with existing antiplatelet drugs, whereby increased potency is offset by elevated bleeding risk. The latest advances in our understanding of thrombus formation have informed the development of new antiplatelet drugs that are potentially safer than currently available therapies.

Platelets are small anucleated cell fragments that ensure the arrest of bleeding after a vessel wall injury. They are also involved in non-hemostatic function such as development, immunity, inflammation, and in the hematogeneous phase of metastasis. While the role of platelets in tumor metastasis has been recognized for 60 years, the molecular mechanism underlying this process remains largely unclear. Platelets physically and functionally interact with various tumor cells through surface receptors including integrins. Platelets express five integrins at their surface, namely α2β1, α5β1, α6β1, αvβ3, and αIIbβ3, which bind preferentially to collagen, fibronectin, laminin, vitronectin, and fibrinogen, respectively. The main role of platelet integrins is to ensure platelet adhesion and aggregation at sites of vascular injury. Two of these, α6β1 and αIIbβ3, were proposed to participate in platelet-tumor cell interaction and in tumor metastasis. It has also been reported that pharmacological agents targeting both integrins efficiently reduce experimental metastasis, suggesting that platelet integrins may represent new anti-metastatic targets. This review focuses on the role of platelet integrins in tumor metastasis and discusses whether these receptors may represent new potential targets for novel anti-metastatic approaches., Competing Interests: The authors declare no conflict of interest.

The ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5β1 and αIIbβ3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5β1 and αIIbβ3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization., (© 2015 by The American Society of Hematology.)

Anti-platelet agents such as aspirin, clopidogrel and antagonists of integrin αIIbβ3 allowed to efficiently reduce morbidity and mortality associated with arterial thrombosis. A major limit of these drugs is that they increase the risk of bleeding. During the last few years, several innovative anti-thrombotic strategies with a potentially low bleeding risk were proposed. These approaches target the collagen receptor glycoprotein (GP) VI, the GPIb/von Willebrand factor axis, the thrombin receptor PAR-1, the activated form of integrin αIIbβ3 or the ADP receptor P2Y1. While an antagonist of PAR-1 was recently marketed, the clinical proofs of the efficiency and safety of the other agents remain to be established. This review evaluates these new anti-platelet approaches toward safer anti-thrombotic therapies., (© Société de Biologie, 2016.)

Background: Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α1β1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown., Methods and Results: Using an in vitro flow assay, we show that laminin-411 (α4β1γ1), laminin-511 (α5β1γ1), and laminin-521 (α5β2γ1), but not laminin-211 (α2β1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis., Conclusions: This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6β1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Objective: The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets. We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIbβ, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIbβ by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation., Approach and Results: We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca(2+) signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)'2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)'2 did not prolong the tail-bleeding time or increase the volume of blood lost., Conclusions: These findings are the first evidence that targeting a subunit other than GPIbα can lead to an antithrombotic effect via the GPIb-V-IX complex. This could represent an alternative way to reduce thrombus formation with a minor impact on hemostasis.

β-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1-/- and β-arr2-/- platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1-/- but not β-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1-/- and β-arr2-/- mice, suggesting no defect in haemostasis. β-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

Objective: The identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, we explored platelet adhesion and activation to tenascin-C (TN-C), a matrix protein preferentially found within atheroma., Methods and Results: We show that platelets efficiently adhere to TN-C under both static and flow conditions. Videomicroscopy revealed a unique behavior under flow, with platelets exhibiting stationary adhesion to TN-C; in contrast, platelets rolled over von Willebrand factor and detached from fibrinogen. Platelet interaction with TN-C was predominantly supported by integrin α(2)β(1) under static conditions, whereas under high shear, it was dependent on both the α(2)β(1) integrin and the glycoprotein Ib-IX complex. Integrin α(IIb)β(3) appeared to play a secondary role but only at low shear rates. The glycoprotein Ib-IX-dependent interaction was indirect, relying on von Willebrand factor, and increased as a function of wall shear rate. Von Willebrand factor bound directly to TN-C, as shown by ELISA and coimmunoprecipitation, suggesting that it acts as a bridge between TN-C and platelets. The adhesion of platelets to TN-C triggered their activation, as demonstrated by a shape change and increases in intracellular calcium level., Conclusions: This study provides evidence that TN-C serves as a novel adhesive matrix for platelets in a context that is relevant to atherothrombosis.

Objective: Adequate hemostasis is extremely important in neurosurgery, commonly requiring the use of topical hemostatic agents. Apart from variable efficacy, the residual presence of these agents may cause foreign body reaction, infection, and delayed bone growth. This study compares the safety and efficacy of commonly used agents with a newly approved agent, Arista (microporous polysaccharide hemospheres; Medafor, Inc., Minneapolis, MN)., Methods: A brain tissue defect was created in 228 Wistar outbred rats, and either no agent (negative control), Arista, Surgicel (oxidized cellulose; Ethicon, Inc., Somerville, NJ), Avitene (microfibrillar collagen; Alcon, Inc., Humacao, PR), FloSeal (gelatin matrix thrombin sealant; Baxter Healthcare Corp., Deerfield, IL), or kaolin (positive control) was implanted. Time to hemostasis was documented. The animals were sacrificed at different intervals up to 28 days, and presence of residual material and foreign body reaction was determined., Results: Arista, Avitene, FloSeal, and Surgicel performed better (defined as complete hemostasis within 1 minute) than control (no treatment). Residual material was not present at any time with Arista, markedly contrasting with the presence of residual material in 100% of lesions in the Avitene, FloSeal, and Surgicel groups on Day 14. Avitene and FloSeal also demonstrated a propensity for causing granuloma formation, whereas Arista and Surgicel showed no such evidence., Conclusion: Each of these hemostatic agents was effective in controlling bleeding in the majority of standardized neurosurgical lesions. Arista degrades more rapidly than Surgicel, Avitene, and FloSeal and does not result in any foreign body reaction.

Thirty-five pediatric patients, 1-16 years of age (median 6.3 years), with neoplastic solid tumors (n=32) or acute leukemia (n=3) underwent peripheral blood stem cell (PBSC) harvest and transplantation at Children's Memorial Hospital between September 1992 and April 1997. A median of four phereses were performed on each patient. Blood samples from 34 of the 35 patients were harvested through existing double-lumen central catheters, using either a Fenwal CS-3000 or COBE Spectra pheresis machine. The pheresis procedures were well tolerated overall. A median of 3.7 x 10(6)/kg CD34+ cells were infused (range, 0.2-15.5 x 10(6)/kg), and all patients engrafted. The median time to an absolute neutrophil count >500/microL was 13 days (range, 9-44 days) and to a platelet count >20,000/microL was 21 days (range, 9-210 days). Two patients died from transplant-related complications. Patients were discharged from the hospital after a median of 22 days (range, 15-64 days). Twenty of the 35 patients are alive, 17 of whom remain disease-free with a median follow-up of 1144 days. According to this study, PBSCs can be successfully harvested and re-infused for marrow reconstitution after myeloablative therapy in children for a variety of pediatric malignancies with low morbidity and mortality.

The present investigation is based on a 2.5 months selbstversuch (self-experiment) of the authors, between October 21 1992, and January 6 1993. 11 healthy students, five females and six males, age 24 to 29 years, and their teachers underwent regular winter swimming at least once a week, for 2 to 10 minutes, at the natural water temperature (6.8 degrees C (October 1992) to 2.0 degrees C (January 1993)) in the southern Baltic Sea. Blood samples were drawn before and 30 and 60 minutes after the cold bath, both at the first and the last day of the swimming season. TSH increased from 0.96 mU/l to 1.42 mU/l (p < 0.01) in the untrained, and from 0.93 mU/l to 1.43 mU/l (p < 0.01) in the cold-trained persons, and decreased thereafter (p < 0.01). Similar changes occurred in cortisol serum concentrations, though psychological stress seemed to interfere with cold stress. Cortisol increased from 99 ng/ml to 133 ng/ml in the untrained, and from 101 ng/ml to 137 ng/ml (p < 0.05) in the cold-trained persons within 30 minutes after cold water immersion, and decreased thereafter (p < 0.01). There were mild decreases in prolactin serum levels after cold stress, whereas FSH, LH and growth hormone remained unaltered. There was a mild initial elevation of serum glucose after cold stress (plus 12 mg/dl, (p < 0.01)) which disappeared after training. There were long term training effects besides the effects on glucose: Basal prolactin levels increased by almost the factor two, and insulin serum levels dropped by almost 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

The production of antibodies to Klebsiella capsular polysaccharides was measured in sera from either HLA-B27-positive (HLA-B27+) or HLA-B27-negative (HLA-B27-) patients with classical ankylosing spondylitis (n = 54). These sera were compared with sera from patients with various rheumatic diseases (n = 82) and HLA-B27+ or HLA-B27- healthy individuals (n = 85). All sera were analyzed by means of an enzyme-linked immunosorbent assay specific to each of the 77 Klebsiella serotypes. The sera from HLA-B27+ patients with ankylosing spondylitis showed a significantly higher antibody frequency to the capsular types K26, K36, and K50 than the sera from HLA-B27- ankylosing spondylitis patients, patients with psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, or reactive arthritis after Yersinia enterocolitica infection, or healthy controls (P < 0.02). The antibodies were of the immunoglobulin G type. No significant antibody response to the other 74 Klebsiella serotypes, noncapsulated mutants of K26, K36, and K50, or preparations of Citrobacter, Serratia, Hafnia, or Morganella spp. or Streptococcus pneumoniae could be detected. The results might suggest a specific association between these capsular types and HLA-B27+ ankylosing spondylitis and might imply their predominance in this disease.

Background: Anticonvulsants have been shown to be effective in many patients with psychiatric disorders, especially those with bipolar affective disorder. We present our clinical experience with divalproex sodium in the treatment of 63 patients with a variety of affective disorders that had proved refractory to conventional pharmacotherapy., Method: We reviewed the charts of 63 patients diagnosed as bipolar I (35 patients), bipolar II (23 patients), or schizoaffective, bipolar type (5 patients). Twelve patients who appeared to have recurrent unipolar depression had a retrospectively recognized history of "covert cycling," with brief periods of socially acceptable hypomania occurring between depressive episodes. Prior to treatment with divalproex, 45 patients had been classified as treatment failures with lithium, 29 patients had been classified as treatment failures with carbamazepine, and 35 patients had also failed on a combination of lithium and carbamazepine. Divalproex was given to these patients and titrated to achieve blood levels in the range of 50 to 100 mg/L., Results: Forty-seven (75%) of the 63 patients responded positively to the addition of divalproex to their regimens. Of 45 patients who had failed to respond to lithium, 38 (84%) responded when divalproex was added. Of 29 patients who had failed to respond to carbamazepine, 20 (69%) responded when divalproex was added. Of 26 rapid-cycling patients, 21 (81%) responded to the addition of divalproex. Side effects required drug withdrawal in 9 patients (14%)., Conclusion: The results confirm previous findings that both mania and rapid mood cycling may respond to a pharmacologic regimen that includes divalproex. Many patients who appear to have recurrent, major unipolar depression may actually be convert cyclers who will respond to divalproex, sometimes in combination with an antidepressant medication.

We report on our clinical experience with a combination of a CNS stimulant (either pemoline or dextroamphetamine) and a monoamine oxidase inhibitor (MAOI) for treating 32 depressed patients (mainly outpatients) refractory to standard antidepressant pharmacotherapy. This combination, though not approved by the FDA, appears to be safe and effective. Twenty-five (78%) of these patients experienced at least 6 months of symptom remission with a stimulant + MAOI combination. Many patients required adjunctive antidepressant treatment, including tricyclics and lithium. Side effects were not excessive, though 6 patients (3 unipolar and 3 bipolar) cycled to mania (N = 1) or hypomania (N = 5). None developed hypertensive crises. With properly motivated and complaint patients and careful clinical monitoring by the prescribing psychiatrist, stimulant potentiation of MAOIs may be a viable option for treatment-resistant depressed patients.

Iatrogenic sexual dysfunction has been associated with many pharmacologic agents. The authors report 6 cases of orgasm dysfunction associated with the use of fluorxetine in 77 depressed outpatients. Fluoxetine is a novel antidepressant known to block the reuptake of serotonin with little effect on other neurotransmitter systems. Because fluoxetine has a specific mechanism of action, it serves as a useful model to hypothesize about potential mechanisms of drug-induced sexual dysfunction. The possible effects of serotonin on central, spinal, and peripheral anatomical areas are discussed in relation to drug-induced sexual dysfunction.