Abstract-This review examines the evidence that platelets play a major role in localizing and controlling the burst of thrombin generation leading to fibrin clot formation. From the first functional description of platelets, it has been recognized that platelets supply factors that support the activation of prothrombin. Studies have demonstrated that on activation, the amount of one specific lipid, phosphatidylserine, is significantly increased on the outer leaflet of platelet membranes. When it was found that phosphatidylserine containing lipid extracts could be substituted for platelets in clotting assays, this suggested the possibility that changes in platelet lipid composition were necessary and sufficient to account for platelet surface thrombin generation. Because a growing body of data suggest that platelet-binding proteins provide much of the specificity for platelet thrombin generation, we review in this report data suggesting that changes in lipid composition are necessary but not sufficient to account for platelet surface regulation of thrombin generation. Also, we review data suggesting that platelets from different individuals differ in their capacity to generate thrombin, whereas platelets from a single subject support thrombin generation in a reproducible manner. Individual differences in platelet thrombin generation might be accounted for by differences in platelet-binding proteins.

Many therapeutic agents are associated with adverse effects in patients. Anticoagulants can engender acute complications such as significant bleeding that increases patient morbidity and mortality. Antidote control provides the safest means to regulate drug action. For this reason, despite its known limitations and toxicities, heparin use remains high because it is the only anticoagulant that can be controlled by an antidote, the polypeptide protamine. To date, no generalizable strategy for developing drug-antidote pairs has been described. We investigated whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Here we show that protein-binding oligonucleotides (aptamers) against coagulation factor IXa are potent anticoagulants. We also show that oligonucleotides complementary to these aptamers can act as antidotes capable of efficiently reversing the activity of these new anticoagulants in plasma from healthy volunteers and from patients who cannot tolerate heparin. This generalizable strategy for rationally designing a drug-antidote pair thus opens up the way for developing safer regulatable therapeutics.

Abstract-The coagulation process has been conceptualized as being primarily dependent on adequate levels of the coagulation proteins. This concept was based on the clear relationship between the bleeding tendency and factor levels in hemophilia. The field is now evolving toward conceptualizing coagulation as being actively regulated by the specialized cellular components of the process. Rather than conceiving coagulation as only a "cascade" of proteolytic reactions, the coagulation reactions occur as overlapping steps on cell surfaces. Components of the old "extrinsic'" and "intrinsic" pathways of coagulation can be thought of as participating in the initiation and propagation of coagulation reactions, respectively. Thus, these pathways are not redundant as they are portrayed in the cascade model, but play distinct and complementary roles. Our understanding of how specific cellular features control the processes of hemostasis and thrombosis is developing rapidly. This review discusses some aspects of the cellular control of coagulation.

FVIIa should be effective in enhancing hemostasis in hypothermic patients. However, because the activity of FVIIa is so dramatically affected by pH, its efficacy may be reduced in acidotic patients.

Recombinant factor VIIa was initially developed for the treatment of hemorrhagic episodes in hemophilic patients with inhibitors to factors VIII and IX. After its introduction, it has also been used "off-label" to enhance hemostasis in nonhemophilic patients who experience bleeding episodes not responsive to conventional therapy. Evidence so far indicates that the use of factor VIIa in hemophilic patients with inhibitors is both safe and effective. Anecdotal reports also suggest that the product is safe and effective in controlling bleeding in nonhemophilic patients. However, its use in these conditions has not been approved by the FDA, and conclusive evidence of its effectiveness from controlled clinical trials is not yet available. Several questions pertaining to the use of factor VIIa require further investigation, including the mechanism of action; the optimal dose; definitive indications; ultimate safety; and laboratory tests for monitoring therapy. (Blood.

No abstract

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Summary Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin‐activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.