ROTEM monitoring of activated and non-activated prothrombin complex concentrate correction of dilutional coagulopathy.

OBJECTIVES: Prothrombin complex concentrates have been used to correct dilutional coagulopathy, but many preparations contain anticoagulants, such as heparin, to counteract their prothrombotic effects. These anticoagulants can interfere with haemostatic assays. The aim of this study was to monitor two different prothrombin complex concentrates for the treatment of albumin dilution in vitro, using rotational thromboelastometry with or without the heparin-antagonising agent protamine. METHODS: Citrated blood from 10 healthy volunteers was, in vitro, diluted 1:1 with 5% albumin and then corrected with a four-factor prothrombin complex concentrate with heparin anticoagulant (Confidex®) corresponding to a clinical dose of 43 IU/kg. Blood samples were tested with or without protamine. An activated prothrombin complex concentrate (APCC) (FEIBA®) without heparin in doses of 50 IU/kg and 100 IU/kg was also tested. Thromboelastometry was performed after recalcification. RESULTS: Albumin dilution significantly affected all thromboelastometry parameters. The four-factor PCC had an additional anticoagulant effect when added to the albumin-diluted blood; it was partially corrected by protamine for all parameters except maximum clot firmness. The APCC significantly improved all parameters, with over-correction of clotting time but only partial correction of maximum clot firmness. CONCLUSIONS: The anticoagulant content of many prothrombin complex concentrates needs to be considered when performing in vitro testing. A heparin-free APCC better corrected an in vitro albumin-induced dilutional coagulopathy than a four-factor PCC, despite of blocking heparin with protamine.

It's "PT" for SCD!

In this issue of Blood, Arumugam et al report that long-term reduction of prothrombin (PT) expression results in diminished vascular inflammation, attenuation of multiple organ damage, and survival advantage in a mouse model of sickle cell disease (SCD).

Genetic diminution of circulating prothrombin ameliorates multiorgan pathologies in sickle cell disease mice.

Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII(lox/-) mice (constitutively carrying ∼10% normal FII) and FII(WT) mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies.

Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma.

Des-γ-carboxy prothrombin (DCP) is a prothrombin precursor produced in hepatocellular carcinoma (HCC). Because of deficiency of vitamin K or γ-glutamyl carboxylase in HCC cells, the 10 glutamic acid (Glu) residues in prothrombin precursor did not completely carboxylate to γ-carboxylated glutamic acid (Gla) residues, leaving some Glu residues remained in N-terminal domain. These prothrombin precursors with Glu residues are called DCPs. DCP displays insufficient coagulation activity. Since Liebman reported an elevated plasma DCP in patients with HCC, DCP has been used in the diagnosis of HCC. Recently, its biological malignant potential has been specified to describe DCP as an autologous growth factor to stimulate HCC growth and a paracrine factor to integrate HCC with vascular endothelial cells. DCP was found to stimulate HCC growth through activation of the DCP-Met-JAK1-STAT3 signaling pathway. DCP might increase HCC invasion and metastasis through activation of matrix metalloproteinase (MMPs) and the ERK1/2 MAPK signaling pathway. DCP has also been found to play a crucial role in the formation of angiogenesis. DCP could increase the angiogenic factors released from HCC and vascular endothelial cells. These effects of DCP in angiogenesis might be related to activation of the DCP-KDR-PLC-γ-MAPK signaling pathway. In this article, we summarized recent studies on DCP in biological roles related to cancer progression and angiogenesis in HCC.

The dual role of innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Antiphospholipid syndrome (APS), as a primary disease or a secondary syndrome in systemic lupus erythematosus (SLE), is characterized by the presence of antiphospholipid antibodies (aPL) and a clinical event. It is likely that both genetic and environmental factors lead to the development of aPL and progression to disease. However, the precise mechanisms are not known. We hypothesize that innate immune activation plays a dual role in APS and SLE, both in the production of aPL (i.e. "initiation" phase) and in the development of a clinical event (i.e. "effector" phase). We have shown that mice immunized with certain phospholipid-binding proteins (e.g. ß2-glycoprotein I (ß2GPI)), plus a concomitant trigger of innate immunity (e.g. a toll-like receptor 4 (TLR4) ligand), produce a strong ß2GPI-reactive T cell response, resulting in high levels of aPL as well as other SLE autoantibodies. We propose that ß2GPI, through its interaction with apoptotic cells, permits B cell epitope spread to multiple SLE autoantibodies. Innate immune activation is also implicated in a murine model of aPL-enhanced thrombus formation. This dual role of innate immune activation provides new insight into the mechanisms involved in the initiation of aPL and other SLE-related autoantibodies, as well as the development of aPL-mediated disease.

Phospholipid-binding proteins differ in their capacity to induce autoantibodies and murine systemic lupus erythematosus.

We have previously shown that immunization of nonautoimmune mice with the phospholipid-binding protein ß2-glycoprotein I (ß2GPI), in combination with lipopolysaccharide (LPS), induces a murine model of systemic lupus erythematosus (SLE), with sequential emergence of autoantibodies and glomerulonephritis. Here, we determine whether the paradigm for induction of murine SLE extends to other phospholipid-binding proteins. Mice were immunized with a phospholipid-binding protein (prothrombin (PT), protein S, or ß2GPI), or a nonphospholipid-binding protein (glu-plasminogen), in the presence of LPS. The breadth and degree of the autoantibody response, and the frequency of glomerulonephritis, varied among the three proteins, with ß2GPI being the most effective in inducing SLE-like disease. The phospholipid-binding proteins also differed in the pattern of serum cytokines they elicited. The most apparent difference between ß2GPI and the other phospholipid-binding proteins was in their ability to bind to LPS: ß2GPI bound to LPS, while PT and protein S did not. Our data suggest that binding to phospholipid(s) is a necessary, but not sufficient, condition for full induction of murine SLE. We propose that other properties, such as physiologic function, avidity for anionic phospholipids, and degree of interaction with other cell surface and/or circulating molecules (particularly LPS) may determine the range and severity of disease.

Effect of Australian elapid venoms on blood coagulation: Australian Snakebite Project (ASP-17).

Snake venoms contain toxins that activate the coagulation network and cause venom-induced consumption coagulopathy. A previously developed mathematical model of the coagulation network was refined and used to describe and predict the time course of changes in the coagulation factors following envenomation by Brown snake (Pseudonaja spp.), Tiger snake (Notechis scutatus), Rough-scaled snake (Tropidechis carinatus) and Hoplocephalus spp. (Stephens banded, Pale headed and Broad headed). Simulations of the time course of the change in coagulation factors were compared to data obtained from a large prospective study of Australian snake bites - the Australian Snakebite Project. The model predictions were also compared against data for partial and complete VICC obtained from the same study. The model simulations were used to understand the differences in consumption and recovery of clotting factors in partial versus complete VICC as well as among bites from different snake types. The model suggested that the venoms were absorbed almost instantaneously and provided a reasonable prediction of the observed concentration of clotting factors over time in patients bitten by Australian elapid snakes. The model predictions suggested a higher consumption of factors (fibrinogen, II and IX in particular) in patients with complete VICC compared to those with partial VICC. The model also predicted that snakes with "Xa-like" venoms may produce a less severe VICC than snakes with "Xa:Va-like" venoms.

Co-occurrence of cardiovascular and prothrombotic risk factors in women with a history of preeclampsia.

OBJECTIVE: Formerly preeclamptic women are at increased risk for remote cardiovascular and thrombotic diseases. We studied co-occurrence of cardiovascular and prothrombotic risk factors within a cohort of formerly preeclamptic women and tested if prevalence of these risk profiles related to onset of preeclampsia in previous pregnancy. METHODS: We evaluated 1,297 nonpregnant formerly preeclamptic women (6-12 months postpartum) for the presence of four risk profiles: circulatory risk profile (hypertension or latent hypertension [low plasma volume, increased vascular resistance, or both]; metabolic syndrome (World Health Organization criteria); thrombophilia (factor V Leiden, prothrombin mutation, or protein C or S deficiency); and hyperhomocysteinemia. Trends between prevalence of these four profiles and onset of preeclampsia were studied using linear regression analysis. RESULTS: After exclusion of 63 women (4.9%) because of incomplete data, 1,234 women were included. One or more risk profiles were detected in 958 of 1,234 (77.6%) formerly preeclamptic women. Circulatory risk profile was more prevalent (66.1%) than hyperhomocysteinemia (18.7%), metabolic syndrome (15.4%), or thrombophilia (10.8%). Prevalence of circulatory risk profile, metabolic syndrome, and hyperhomocysteinemia decreased significantly with gestational age at delivery, whereas thrombophilia did not (P=.22). There was minimal overlap (less than 2%) between metabolic syndrome, thrombophilic profile, and hyperhomocysteinemia. CONCLUSION: Circulatory risk profile is present in two thirds of formerly preeclamptic women. Metabolic syndrome, thrombophilia, and hyperhomocysteinemia are prevalent in 10-20%. There is considerable overlap between circulatory risk profile and other profiles, but not among the three other profiles. Prevalence of these risk factors, except thrombophilia, decreases with gestational age at delivery in preceding pregnancy. LEVEL OF EVIDENCE: : II.

Role of microparticles in sepsis.

This review discusses the role of microparticles in inflammation, coagulation, vascular function, and most importantly, their physiological and pathological functions in sepsis. Microparticles are proinflammatory, procoagulant membrane vesicles released from various cell types. They are detectable in normal individuals and basal levels correlate with a balance between cell proliferation, stimulation, and destruction. Haemostatic imbalance leads to various pathological states of inflammation and thrombosis including cardiovascular disease and sepsis, where circulating microparticles display both an increase in number and phenotypic change. Microparticles, mainly of platelet origin enable both local and disseminated amplification of the haemostatic response to endothelial injury through exposure of phosphatidylserine, tissue factor, and coagulation factor binding sites. Surface expression of membrane antigens by microparticles facilitates cytoadhesion, chemotaxis, and cytokine secretion to drive a proinflammatory response. Microparticles behave as vectors in the transcellular exchange of biological information and are important regulators of endothelial function and angiogenesis. The extent to which circulating microparticles contribute to the pathogenesis of sepsis and disseminated intravascular coagulation is currently unknown. Microparticles may in fact be beneficial in early sepsis, given that activated protein C bound to endothelium-derived microparticles retains anticoagulant activity, and increased circulating microparticles are protective against vascular hyporeactivity. Elevated levels of microparticles in early sepsis may therefore compensate for the host's systemic inflammatory response. Importantly, in vivo, septic microparticles induce deleterious changes in the expression of enzyme systems related to inflammation and oxidative stress, thus they may represent important contributors to multi-organ failure in septic shock.

Hepatocellualar carcinoma serum markers.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in some areas of the world. In most cases, HCC is diagnosed at a late stage. Therefore, the prognosis of patients with HCC is generally poor. The recommended screening strategy for patients with cirrhosis includes the determination of serum α-fetoprotein (AFP) levels and an abdominal ultrasound every 6 months to detect HCC at an earlier stage. AFP, however, is a marker characterized by poor sensitivity and specificity, and abdominal ultrasound is highly dependent on the operator's experience. In addition to AFP, Lens culinaris agglutinin-reactive AFP (AFP-L3), des-γ-carboxy prothrombin (DCP), glypican-3 (GPC-3), osteopontin (OPN), and several other biomarkers (such as squamous cell carcinoma antigen-immunoglobulin M complexes [SCCA-IgM], alpha-1-fucosidase [AFU], chromogranin A [CgA], human hepatocyte growth factor, insulin-like growth factor) have been proposed as markers for the early detection of HCC. For these markers, we describe the mechanisms of production, and their diagnostic and prognosis roles. None of them is optimal; however, when used together, their sensitivity in detecting HCC is increased. Recent research has shown that some biomarkers have mitogenic and migratory activities in the angiogenesis of HCC and are a factor of tumor growth.

PIVKA-II plasma levels as markers of subclinical vitamin K deficiency in term infants.

BACKGROUND: As the vitamin K content of human milk is low and the newborn infant's stores of vitamin K are small, vitamin K deficiency with hemorrhage in the newborn is a worldwide problem. Proteins Induced by Vitamin K Absence (PIVKA-II) are the inactive under-γ-carboxylated forms of vitamin K-dependent clotting factors and they could be useful in predicting subclinical vitamin K deficiency (VKD). OBJECTIVES: To demonstrate that PIVKA-II are earlier markers of subclinical VKD than Prothrombin time (PT) in exclusively breast-fed newborns. METHODS: A prospective, controlled, randomized study, including 53 term newborns receiving vitamin K prophylaxis (0.5 mg i.m.) at birth, was performed. At 30 days newborns were divided into three groups (G) receiving respectively: 25 µg/die of vitamin K (G I), 12 µg/die (G II) or placebo (G III). PIVKA-II and PT were measured on 30th and 90th days of life. RESULTS: G III and GII showed a significant increase in PIVKA-II from 30 to 90 days of life respectively from 2.6 to 4.7 (p = 0.001) and from 2.3 to 3.5 (p < 0.001). No significant changes were found in GI. PT showed no significant changes among groups. CONCLUSIONS: PT is a less sensitive marker than PIVKA II. Oral supplementation with 25 µg/die avoids an increase of PIVKA-II. Despite increased PIVKA-II do not mean an impending occurrence of bleeding, they highlight a subclinical VKD and its relative risk.

Vascular behcet and mutations in thrombogenic genes: methylene tetrahydrofolate reductase, factor V, and prothrombin.

Vasculitis, thrombophlebitis, arterial aneurysms, and occlusions occur in about 25% of patients with Behçet's disease (BD). The common inherited gene defects, factor V (FV) 1691A (Leiden), methylene tetrahydrofolate reductase (MTHFR) 677T, and prothrombin 20210A, are known risk factors for thrombosis. The aim of the study was to evaluate the contribution of these mutations to thrombosis in Israeli patients with BD. Fifty-four patients with BD (n=54; 27 men and 27 women) underwent clinical and genetic evaluation. Most patients (n=43; 79.6%) were of Arab descent (31 sporadic and 12 familial cases from 4 families), and 11 patients (20.4%) were of Jewish descent (all sporadic cases). The FV Leiden mutation was identified in five patients (9.2%), and eight patients were MTHFR 677TT homozygotes (14.8%). None had the 20210A mutant prothrombin allele. No statistical differences between carriers and noncarriers with regards to demographic and disease manifestations were calculated. Arabs were diagnosed earlier than Jewish patients (25.8±11.6 compared with 37.2±10.7, p=0.01, respectively), but Jewish patients had, respectively, more events of deep vein thrombosis (DVT) compared with Arabs (3 of 11, 27.3% and 3 of 43, 7%, p=0.09). Thrombotic events in our patients with BD were not associated with variations in thrombophilic genes.

Activation of factor XI by products of prothrombin activation.

The prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to ß-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by ß-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, ß-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

Method for in vitro analysis of nanoparticle thrombogenic properties.

Thrombus formation is complex process involving both cellular and molecular (protein) components. Platelets are responsible for maintaining hemostasis and for preventing excessive bleeding. These cells aggregate along with other plasma components and blood cells to form blood clots. Undesirable platelet aggregation may lead to life-threatening conditions such as stroke. Thrombogenicity is the property of a material to induce the formation of a thrombus, which results in partial or complete occlusion of a blood vessel. The tendency to cause platelet aggregation and perturb plasma coagulation can serve as an in vitro measure of a nanomaterial's likelihood to be thrombogenic in vivo. This chapter describes a procedure for in vitro analyses of platelet aggregation and plasma coagulation time. Platelet-rich plasma (PRP) is obtained from freshly derived human whole blood and incubated with nanoparticles. Then the plasma is examined using a particle count and size analyzer to determine the number of active platelets. The percent aggregation is calculated by comparing the number of active platelets in the nanoparticle-exposed sample to control plasma. To measure the plasma coagulation time, platelet-poor plasma from human whole blood is exposed to nanoparticles in vitro and analyzed in prothrombin (PT), activated partial thromboplastin (APTT), and thrombin time assays.

NSA9, a human prothrombin kringle-2-derived peptide, acts as an inhibitor of kringle-2-induced activation in EOC2 microglia.

In neurodegenerative diseases, such as Alzheimer's and Parkinson's, microglial cell activation is thought to contribute to CNS injury by producing neurotoxic compounds. Prothrombin and kringle-2 increase levels of NO and the mRNA expression of iNOS, IL-1beta, and TNF-alpha in microglial cells. In contrast, the human prothrombin kringle-2 derived peptide NSA9 inhibits NO release and the production of pro-inflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in LPS-activated EOC2 microglia. In this study, we investigated the anti-inflammatory effects of NSA9 in human prothrombin- and kringle-2-stimulated EOC2 microglia. Treatment with 20-100 muM of NSA9 attenuated both prothrombin- and kringle-2-induced microglial activation. NO production induced by MAPKs and NF-kappaB was similarly reduced by inhibitors of ERK (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), and NSA9. These results suggest that NSA9 acts independently as an inhibitor of microglial activation and that its effects in EOC2 microglia are not influenced by the presence of kringle-2.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

[Influence of prothrombin cleavage products on platelet activation and aggregation].

Meizothrombin efficiently activates mechanically triggered platelets and enhance collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma. Thus, in clotting system activation zone meizothrombin is able to enhance process of involving platelets in clotformation. Pre-thrombin 1 inhibits collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma and so regulates not only plasm but platelet hemostasis by reverse negative relation.

Des-gamma-carboxy prothrombin increases the expression of angiogenic factors in human hepatocellular carcinoma cells.

AIMS: Des-gamma-carboxyl prothrombin (DCP) is a serum protein produced by hepatocellular carcinoma (HCC) cells. The aim of this study was to evaluate the angiogenic activity of DCP in HCC cells. MAIN METHODS: The proliferation of HCC cells was measured by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. The growth of HCC cells was also evaluated in vivo by using the xenografts in nude mice. The enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of angiogenic factors in supernatant of cell culture. The expression of angiogenic factors was examined by Western blot analysis and immunohistochemical staining. KEY FINDINGS: DCP displayed the stimulation of HCC cell growth in a dose (5-80 ng/ml) and time (24-96 h) dependent manner. The increase of cell growth was also observed in nude mice bearing well-established, palpable HepG2 and SMMC-7721 xenografts after 2 weeks administration of DCP. HCC cell growth was accompanied by the elevated levels of angiogenic factors. The levels of vascular endothelial growth factor (VEGF), transforming growth factor-alpha (TGF-alpha) and basic fibroblast growth factor (bFGF) in supernatant of SMMC-7721 cells were increased from 47, 126, and 60 pg/10(6) cells/24 h to 400, 208, and 298 pg/10(6) cells/24 h, respectively, after 72 h incubation with 80 ng/ml of DCP. The results of Western blot analysis and immunohistochemical staining of HCC xenografts also showed the significant increase of VEGF, TGF-alpha, and bFGF in HCC cells. SIGNIFICANCE: These results provide the information that DCP is a type of growth factor in progression of HCC.

Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases.

Emerging evidence demonstrates that thrombin exerts physiological and pathological functions in the central nervous system. Both prothrombin and its active form thrombin have been detected locally in the brain. The cellular functions of thrombin are mainly regulated by G protein-coupled protease-activated receptors (PARs). Thrombin can signal via PAR-1, PAR-3 and PAR-4. Some neurological diseases (e.g. Alzheimer's disease or Parkinson's disease) are characterized by increased levels of both active thrombin and PAR-1. This indicates that thrombin and its receptor may be closely involved in the development of neurodegenerative processes. The role of thrombin in brain injury can be either protective or deleterious, depending on the concentration of thrombin. Thrombin at high concentrations exacerbates brain damage. In contrast, low concentrations of thrombin rescue neural cells from death after brain insults. Also thrombin preconditioning has neuroprotective effects. Therefore, thrombin and thrombin receptors represent novel therapeutic targets for treating neurodegenerative diseases.