Pieter H. Reitsma

New Fundamentals in Hemostasis

Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.

Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect

The relevance of heterozygosity for hereditary protein C deficiency as a risk factor for venous thrombosis has been disputed because heterozygotes without symptoms have been identified among blood donors and relatives of homozygotes. As a result, clinicians do not know whether to offer prophylaxis or not. We have compared thrombosis-free survival in 161 heterozygous and normal members of the families of 24 heterozygotes for protein C deficiency referred from several centres in the Netherlands and with a history of symptoms. We studied the influence of heterozygosity and of putative additional risk factors on the occurrence of thrombotic events noted when a medical history was taken. Protein C activities were measured but a diagnosis of heterozygosity was based on the presence of the specific mutation in one of the protein C genes identified in the proband of the family. We found a significant difference in the thrombosis-free survival of the 77 heterozygotes and 84 normals: by age 45, 50% of heterozygotes and 10% of normal relatives can be expected to have had a manifestation of venous thromboembolism. The presence of such a mutation was clearly associated with an increased risk of venous thrombotic events. Thrombotic events occurred more often in years in which the patient had been immobile for more than a week or had had surgery. Other putative risk factors showed no significant effect in the incidence of thrombotic events. About 50% of all first episodes and 65% of recurrences of venous thromboembolism in the heterozygotes were spontaneous--ie, there was no predisposing event such as surgery or pregnancy. There was no increased risk for arterial occlusions in heterozygotes. We conclude that members of the family of a symptomatic heterozygote proband who are heterozygous for the mutation in the protein C gene have an increased risk of venous thrombotic events compared with their normal family members. For such individuals prophylactic anticoagulation should be considered; the decision will need to be taken on an individual basis.

Gene variants associated with deep vein thrombosis.

CONTEXT The genetic causes of deep vein thrombosis (DVT) are not fully understood. OBJECTIVE To identify single-nucleotide polymorphisms (SNPs) associated with DVT. DESIGN, SETTING, AND PATIENTS We used 3 case-control studies of first DVT. A total of 19 682 gene-centric SNPs were genotyped in 443 cases and 453 controls from the Leiden Thrombophilia Study (LETS, 1988-1992). Twelve hundred six SNPs associated with DVT were reinvestigated in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis study (MEGA-1, 1999-2004) in a subset of 1398 cases and 1757 controls. Nine SNPs associated with DVT in both LETS and MEGA-1 were investigated a third time in 1314 cases and 2877 controls from MEGA-2, a second subset of MEGA. Additional SNPs close to one SNP in CYP4V2 were genotyped in LETS and MEGA-1. MAIN OUTCOME MEASURE Odds ratios (ORs) for DVT were estimated by logistic regression. False discovery rates served to investigate the effect of multiple hypothesis testing. RESULTS Of 9 SNPs genotyped in MEGA-2, 3 were strongly associated with DVT (P < .05; false discovery rate < or =.10): rs13146272 in CYP4V2 (risk allele frequency, 0.64), rs2227589 in SERPINC1 (risk allele frequency, 0.10), and rs1613662 in GP6 (risk allele frequency, 0.84). The OR for DVT per risk allele was 1.24 (95% confidence interval [95%CI], 1.11-1.37) for rs13146272, 1.29 (95% CI, 1.10-1.49) for rs2227589, and 1.15 (95% CI, 1.01-1.30) for rs1613662. In the region of CYP4V2, we identified 4 additional SNPs (in CYP4V2, KLKB1, and F11) that were also associated with both DVT (highest OR per risk allele, 1.39; 95% CI, 1.11-1.74) and coagulation factor XI level (highest increase per risk allele, 8%; 95% CI, 5%-11%). CONCLUSIONS We identified SNPs in several genes that were associated with DVT. We also found SNPs in the region around the SNP in CYP4V2 (rs13146272) that were associated with both DVT and factor XI levels. These results show that common genetic variation plays an important role in determining thrombotic risk.

Variants of Toll-Like Receptor 4 Modify the Efficacy of Statin Therapy and the Risk of Cardiovascular Events

Background—Atherosclerosis is increasingly considered to be a chronic inflammatory process. We examined whether genetic variants of the toll-like receptor 4 (TLR4), which are correlated with impaired innate immunity and with progression of carotid atherosclerosis, are also associated with coronary atherosclerosis and predict the risk of cardiovascular events. Methods and Results—Two polymorphisms of the TLR4 gene (Asp299Gly and Thr399Ile) were determined in 655 men with angiographically documented coronary atherosclerosis. All patients participated in a prospective cholesterol-lowering trial evaluating the effect on coronary artery disease and were randomly assigned to either pravastatin or placebo for 2 years. There were no significant differences between genetically defined subgroups with respect to baseline risk factors, treatment, or in-trial changes of lipid, lipoprotein, or angiographic measurements. Genotype was not associated with progression of atherosclerosis. In the pravastatin group, 299Gly carriers had a lower risk of cardiovascular events during follow-up than noncarriers (2.0% versus 11.5%, P =0.045). Among noncarriers, pravastatin reduced the risk of cardiovascular events from 18.1% to 11.5% (P =0.03), whereas among 299Gly carriers this risk was strikingly reduced from 29.6% to 2.0% (P =0.0002, P =0.025 for interaction). Conclusions—Among symptomatic men with documented coronary artery disease, the TLR4 Asp299Gly polymorphism was associated with the risk of cardiovascular events. This variant also modified the efficacy of pravastatin in preventing cardiovascular events, such that carriers of the variant allele had significantly more benefit from pravastatin treatment.

Postoperative ileus is maintained by intestinal immune infiltrates that activate inhibitory neural pathways in mice.

BACKGROUND AND AIMS Postoperative ileus after abdominal surgery largely contributes to patient morbidity and prolongs hospitalization. We aimed to study its pathophysiology in a murine model by determining gastric emptying after manipulation of the small intestine. METHODS Gastric emptying was determined at 6, 12, 24, and 48 hours after abdominal surgery by using scintigraphic imaging. Intestinal or gastric inflammation was assessed by immune-histochemical staining and measurement of tissue myeloperoxidase activity. Neuromuscular function of gastric and intestinal muscle strips was determined in organ baths. RESULTS Intestinal manipulation resulted in delayed gastric emptying up to 48 hours after surgery; gastric half-emptying time 24 hours after surgery increased from 16.0 +/- 4.4 minutes after control laparotomy to 35.6 +/- 5.4 minutes after intestinal manipulation. The sustained delay in gastric emptying was associated with the appearance of leukocyte infiltrates in the muscularis of the manipulated intestine, but not in untouched stomach or colon. The delay in postoperative gastric emptying was prevented by inhibition of intestinal leukocyte recruitment. In addition, postoperative neural blockade with hexamethonium (1 mg/kg intraperitoneally) or guanethidine (50 mg/kg intraperitoneally) normalized gastric emptying without affecting small-intestinal transit. The appearance of intestinal infiltrates after intestinal manipulation was associated with increased c-fos protein expression in sensory neurons in the lumbar spinal cord. CONCLUSIONS Sustained postoperative gastroparesis after intestinal manipulation is mediated by an inhibitory enterogastric neural pathway that is triggered by inflammatory infiltrates recruited to the intestinal muscularis. These findings show new targets to shorten the duration of postoperative ileus pharmacologically.

Activation of Inflammation and Coagulation After Infusion of C-Reactive Protein in Humans

C-reactive protein (CRP) has been postulated to play a causal part in atherosclerosis and its acute complications. We assessed the effects of CRP-infusion on coagulation and inflammatory pathways to determine its role in atherothrombotic disease. Seven male volunteers received an infusion on two occasions, containing 1.25 mg/kg recombinant human CRP (rhCRP) or diluent, respectively. CRP-concentrations rose after rhCRP-infusion from 1.9 (0.3 to 8.5) to 23.9 (20.5 to 28.1) mg/L, and subsequently both inflammation and coagulation were activated. This sequence of events suggests that CRP is not only a well known marker of cardiovascular disease, but is also probably a mediator of atherothrombotic disease.

The relationship between tissue factor and cancer progression: insights from bench and bedside

It is now widely recognized that a strong correlation exists between cancer and aberrant hemostasis. Patients with various types of cancers, including pancreatic, colorectal, and gastric cancer, often develop thrombosis, a phenomenon commonly referred to as Trousseau syndrome. Reciprocally, components from the coagulation cascade also influence cancer progression. The primary initiator of coagulation, the transmembrane receptor tissue factor (TF), has gained considerable attention as a determinant of tumor progression. On complex formation with its ligand, coagulation factor VIIa, TF influences protease-activated receptor-dependent tumor cell behavior, and regulates integrin function, which facilitate tumor angiogenesis both in vitro and in mouse models. Furthermore, evidence exists that an alternatively spliced isoform of TF also affects tumor growth and tumor angiogenesis. In patient material, TF expression and TF cytoplasmic domain phosphorylation correlate with disease outcome in many, but not in all, cancer subtypes, suggesting that TF-dependent signal transduction events are a potential target for therapeutic intervention in selected types of cancer. In this review, we summarize our current understanding of the role of TF in tumor growth and metastasis, and speculate on anticancer therapy by targeting TF.

IL-8 plasma concentrations and the risk of future coronary artery disease in apparently healthy men and women - The EPIC-Norfolk prospective population study

Objective—To study the role of IL-8 in predicting future coronary artery disease (CAD) in apparently healthy men and women. Methods and Results—A nested case-control study was performed in the prospective EPIC-Norfolk population study. We measured baseline IL-8 concentrations among 785 apparently healthy individuals in whom fatal or nonfatal CAD developed during follow-up and 1570 matched controls. Baseline IL-8 concentrations were higher in cases than in matched controls (3.5 pg/mL versus 3.1 pg/mL, P = 0.001). The risk of future CAD increased with increasing quartiles of IL-8 (P linearity < 0.0001). Among individuals in the highest IL-8 quartile, the unadjusted odds ratio for future CAD was 1.72 (95% CI, 1.34 to 2.21; P < 0.0001). The odds ratio for future CAD was still significant after adjustment for traditional risk factors (OR, 1.58; 95%CI, 1.19 to 2.09; P = 0.002) and after additional adjustment for C-reactive protein and white cell count (OR, 1.77; 95% CI, 1.21 to 2.60; P = 0.001). Conclusions—We conclude that among apparently healthy men and women, elevated levels of IL-8 are associated with an increased risk of future CAD. These prospective data support a role for IL-8 in the development of CAD events.

Mechanistic View of Risk Factors for Venous Thromboembolism

Venous thromboembolism is an episodic disease with an annual incidence of 2 to 3/1000 per year that is associated with a high morbidity and mortality. Risk factors for venous thromboembolism come in many guises. They fit into an extended version of Virchows triad and they tilt the hemostatic balance toward clot formation. This can be achieved by decreasing blood flow and lowering oxygen tension, by activating the endothelium, by activating innate or acquired immune responses, by activating blood platelets, or by increasing the number of platelets and red blood cells or modifying the concentrations of pro- and anticoagulant proteins in the blood. In this narrative review we will discuss the known common risk factors within this pathophysiological framework.

Genotypic Variation in the Promoter Region of the Protein C Gene Is Associated With Plasma Protein C Levels and Thrombotic Risk

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals with protein C deficiency are at risk for thrombophlebitis, deep-vein thrombosis, and pulmonary embolism. Genetic analysis of a number of randomly chosen healthy individuals revealed three polymorphisms, C/T at -654, A/G at -641, and A/T at -476, in the protein C promoter region. To investigate whether these genetic variations associate with the plasma protein C level, we determined the genotype for the three polymorphisms and measured plasma protein C levels in 240 individuals not deficient in protein C. The mean protein C level of these individuals was 103%. Interestingly, individuals with the homozygous CGT genotype (n = 40) had a mean protein C level of 94%, whereas individuals with a homozygous TAA genotype (n = 28) had a mean protein C level of 116%. This difference in mean protein C levels between the CGT and TAA groups (P < .001) could not be explained by environmental factors known to influence protein C levels in the normal population. Plasma factor II and factor X levels did not differ between the two groups, which makes a difference in liver function an unlikely cause. Finally, we tested whether the genotype associated with lower protein C levels is associated with higher thrombotic risks. This analysis showed that compared with the genetic variant associated with higher protein C levels (TT/AA/AA), the genetic variant associated with lower protein C levels (CC/GG/TT genotype) is indeed a risk factor for thrombosis (OR, 1.6; 95% confidence interval, 1.0 to 2.5).