An ELISA assay for quantitation of the thrombin-heparin cofactor II complex (T-HC II) in plasma was developed. Plasma was incubated with immobilized, specific antibodies to human thrombin. The second, biotinylated antibody was directed against human HC II. The assay was insensitive to thrombin-antithrombin complex (TAT) and to uncomplexed HC II. In plasma samples from 31 normal individuals (aged 21-68, mean 43.3 years), the T-HC II ranged 0.3-6.1 ng/ml; median 1.5, mean 2.0, and SD 1.6 ng/ml. In plasma samples from 13 patients with disseminated intravascular coagulation (DIC), T-HC II ranged 0.4-30.0 (median 13.5) ng/ml. In plasma samples from 6 patients in which the clinical suspicion of DIC was not verified, T-HC II complex ranged 1.4-14.3 (median 3.6) ng/ml. In plasma samples with elevated T-HC II levels, TAT was usually elevated, and on the average more than was T-HC II. These results indicate that HC II contributes significantly to the inactivation of in vivo generated thrombin.

The well-known coagulation inhibitors antithrombin and protein C, and the more recently described inhibitors, heparin cofactor II and extrinsic pathway inhibitor, were measured in plasma during a 7-day observation period, from patients with pneumonia (n = 13), and in stroke patients with infarction (n = 9) and haemorrhage (n = 9). In patients with pneumonia, elevated fibrinopeptide A levels and subnormal antithrombin and protein C levels suggested some degree of consumption of the inhibitors. Later, an increase was observed for all the inhibitors, but was most conspicuous for heparin cofactor II which reached high normal values. C-reactive protein, initially markedly elevated, decreased rapidly. This finding suggests that heparin cofactor II might act as a delayed acute phase reactant. In stroke patients only small, not statistically significant, changes occurred during the observation period, except for heparin cofactor II which increased in patients with haemorrhagic stroke.

Summary Extrinsic coagulation pathway inhibitor may be an important regulator of haemostasis to prevent thrombosis after tissue damage. The functional activity of this inhibitor was determined using a chromogenic substrate assay, and compared to the activities of anti thrombin, heparin cofactor II and protein C during the perioperative period of elective hip replacement (n = 28), cholecystectomy (n = 11), and vascular surgery (n = 5). Peroperatively, all the inhibitors decreased rather similarly and to the same degree as the decrease in albumin concentration. The decreases during hip surgery were about 2-fold the decreases observed during cholecystectomy. A significant peroperative increase in extrinsic pathway inhibitor activity was observed in vascular surgery, probably due to a bolus injection of heparin. Antithrombin, heparin cofactor II and protein C levels normalized on days 3-5 postoperatively in all three patient groups. Sustained low levels of extrinsic pathway inhibitor were observed on postoperative days 1 to 7 in hip surgery patients. Apparently, extrinsic pathway inhibitor is not an acute phase reactant. In uncomplicated surgery, the decreases of the coagulation inhibitor levels are mainly due to hemodilution.

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCIIOslo) from the plasma of ne of these individuals. HCIIOslo or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G----A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.

Serial determinations of plasma coagulation inhibitor levels were performed with chromogenic substrate activity assays in 7 patients with cancer. At time of diagnosis normal median activities of Antithrombin, Protein C, Heparin Cofactor II and Extrinsic Pathway Inhibitor were found. The inhibitor activities changed significantly with the progress of malignant disease; Antithrombin, Protein C and Heparin Cofactor II decreased whereas Extrinsic Pathway Inhibitor increased. Determinations in 13 additional patients in the terminal phase of cancer confirmed this finding. The inhibitor activities were expressed in per cent of a pooled reference plasma. In the total series of 20 patients studied, median activity of Extrinsic Pathway Inhibitor was 183% (range 61-378%) and significantly (p less than 0.005) above age-adjusted normal reference 10 days (range 1-20 days) prior to death. Median activities of Antithrombin was 59% (range 20-109%), of Protein C 54% (range 24-130%) and Heparin Cofactor II 59% (range 33-110%), all significantly below age adjusted normal reference (p less than 0.001). The coagulation inhibitor levels seem related to the stage of disease in patients with cancer.

The plasma inhibitor(s) of factor VIIa-tissue thromboplastin cooperates with factor Xa. This "Extrinsic Pathway Inhibitor" has been quantitated with a sensitive chromogenic substrate assay. Gel filtration of plasma separates 3 EPI peaks. Postoperatively, both EPI and the other coagulation inhibitors decrease. Unlike the other inhibitors, EPI is usually normal in severe liver cirrhosis. In disseminated intravascular coagulation, EPI levels vary considerably.

ABSTRACT. The coagulation inhibitors heparin cofactor II (HCII), antithrombin (AT) and protein C (PC) were measured in healthy term and preterm infants in order to establish reference standards. The mean value for HCII in term infants was found to be about half of the adult values. Values below 25% in healthy infants may suggest hereditary deficiency states. One girl with congenital HC II deficiency was detected. Mean AT and PC levels were somewhat higher than HC II. Healthy preterm infants have significantly lower HC II and AT values than healthy term infants. Serial AT measurements have been used in monitoring seriously ill infants and used as a prognostic indicator. In a small number of unhealthy neonates HC II was reduced to an even greater extent than AT, and on recovery normalized more rapidly than AT.

The coagulation inhibitor heparin cofactor II (HC II) was measured in various liver diseases and compared with antithrombin (AT), Normotest (NT), albumin and bilirubin. The lowest mean HC II level was found in alcoholic cirrhosis and the reduction reflected the degree of liver failure. A statistically significant association was found between HC II and AT (r = 0.79), NT (r = 0.71) and albumin (r = 0.66) (P less than 0.001), and there was a negative association between HC II and bilirubin (r = -0.55, P less than 0.001). HC II values below 42% in alcoholic cirrhosis seem to indicate a poor prognosis, since nine out of 18 patients with such recordings died. In contrast, no association between mortality and AT, NT and albumin levels was observed. In conclusion, HC II seems to be a reliable liver function test. It may serve as a prognostic indicator and in that respect it may be superior to NT and AT. The site of production is most probably the hepatocyte.

In our normal material of 379 blood donors,3 indivi-uals had values below mean −2.5 SD (below 56%).Further studies revealed hereditary deficiency in two of these individuals. In the family study,5 out of 7 individuals had heparin cofactor II (HC II) values below 56%. In only one of these 5 a history of DVT was obtained. In deceased members of her family, however, frequent episodes of thromboembolic disease had occurred.Crossed immunoelectrophoresis (CIE) was performed in plasma from deficiency individuals from both families. Heparin in the first dimension gave a pattern similar to that observed with normal pooled plasma. The inactivation of thrombin by HC II is preferentially accelerated by dermatan sulfate (DS).HC II consumption in in vitro coagulation is increased by DS rather than by heparin. This prompted the addition ofTBS to the first dimension, which makes the antigen move faster, but produced no alteration in the antigenic pattern or size, neither in pooled plasma nor in a family member with normal activity. In family members with low HC II activity, DS in the first dimension, resulted in an abnormal CIE pattern with two distinct precipitation arcs with identity pattern. Molecular heterogeneity of HC II has previously not been reported.Screening 70 individuals who had sustained thrombosis before the age of 50,no values suggesting congenital deficiency were encountered.Conclusion. The CIE findings suggest that plasma of affected family members contain two types of HC II molecules of immunological identity. One type shows the normal accelerated mobility in the presence of DS and the other lacks this effect. The abnormal electrophoretic pattern becomes, however, first apparent when DS is added to the first dimension. When looking for molecular defects in congenital HC II deficiency, it is important that the first dimension is run with DS.

The coagulation inhibitors heparin cofactor II (HC II), protein C (PC) and antithrombin (AT) were measured in 14 patients undergoing uncomplicated abdominal, orthopedic or vascular surgery. Marked transient decrease of all the inhibitors was found after all surgical procedures, most prominently in vascular surgery. The greatest mean reduction was in HC II. The fall in PC and AT showed a pattern similar to that of albumin. In contrast to some earlier suggestions our results indicated that, except for HC II, the reduction probably was not due to consumption. Redistribution, hemodilution and, probably, reduced synthesis were the most important contributing factors. Serial measurements of the three coagulation inhibitors may be of prognostic value, but the present study's observed mean decrease of c. 25% in abdominal and 50-62% in vascular surgery must be borne in mind. Still lower inhibitor levels suggest consumption and a complicated postoperative course.

Hypercoagulation after surgical trauma is probably induced by tissue thromboplastin (TP) released into the circulation. EPI, in conjuction with activated factor x (FXa), is a potent inhibitor of the IP-factor VII complex. EPI levels (chromogenic substance assay) were compared to other inhibitors; antithrombin (AT), heparin cofactor II (HCII), andprotein C (PC) in patients undergoing cholecystectomy(n=4), hip prosthesis operation (n=5),and aortic grafts operationa(n=5) Mean AT and PC levels parallelled the decrease in albumin. HCII decreased more suggesting a real consumption ofsurgery. The changes in EPI levels depended on the type ofsurgery. In hip surgery, the decline in EI levels was marked and parallelled HCII. In constrast to the other inhibitors, EPI levels stayed low over the first week after surgery: In cholecystectomy, changes were less marked and all inhibitors behaved similar. During aortic operations, EPI increased from mean 110% preoperatively to 252% peroperatively. It decreased to 86% the first day after operation. It then increased similar to the other inhibitors, but the level stayed higher than expected from the preoperative value. The patients received 3000 IU heparin peroperatively. In conclusion, hip operations produce a sustained drop in EPI activity.In aortic operations injection of heparin induced a dramatic, shortlived increase peroperatively followed by a restoration to high normal levels.

The activity levels of the “new” coagulation inhibitors, heparin cofactor II (HC II) and extrinsic pathway inhibitor (EPI),have been determined with chromogenic substrates assays, in patients with pneumonia (n=8), disseminated intravascular coagulation (DIC) (n=8) and various liver diseases (n=19). For comparison antithrombin (AT) and Protein C (PC) were also measured. In cases with DIC low values (<50%) for HC II,AT and PC were found, while EPI showed a much greater variation (60-190%). Persistent low values heralds a poor prognosis.In survivors is rapidly normalized. In pneumonia , initially low levels (except HC II),were normalized on day 7. HC II may be an acute phase reactant. Conclusion.In cirrhosis, subnormal HC II values suggests reduced synthesis.High EPI values in cirrhosis suggests extrahepatic synthesis.The mechanisms for reduced HC II in DIC,might besides consumption and reduced synthesis, be the liberation of dermatan sulfate from injured intima with increased consumption. Changes in HC II,AT and PC are similar, whereas EPI seems to have different production and metabolism

Protein C in citrated plasma is found to be specifically activated by the snake venom derivative Protac, and the activator is used in a simple, automated assay method. The activation is completed in less than 120 s and an isolation of protein C from its inhibitor before activation is not necessary. The activated protein C is determined with the chromogenic substrate S-2366. Therapeutic concentrations of heparin in the test sample do not influence the result. A strong positive correlation to immunoassay of protein C was found (r = 0.92). Three cases of probable hereditary protein C deficiency belonging to the same family were discovered during the study.

In vitro coagulation produces a consumption of heparin co factor II (HC II) of 8–10% both in plasma from normal individuals and patients whereas antithrombin (AT) consumption ranges 40–50%.In blood from heparin treated patients consumption is similar or greater. In blood from warfarin treated patients, consumption is decreased. Addition of heparin prior to clotting has little effect on HC II consumption, but high heparin concentration reduces AT consumption. Addition of dermatan sulfate has no effect on AT consumption, but increases HC II consumption dramatically. In consumption coagulopathy, the HC II levels are as low as AT, possibly reflecting intravascular consumption accelerated by vascular glycosaminoglycans.

In response to an article on the effect of contraceptive agents on antithrombin III the role of other coagulation inhibitors in inducing thrombosis is stressed namely protein C and heparin cofactor II. Deficiency of both protein C and heparin cofactor II leads to deep venous thrombosis. In 10 to 15% of deep venous thrombosis cases deficiency of antithrombin or protein C was the underlying cause according to research data. Congenital protein C deficiency is more frequent than antithrombin deficiency. Protein C levels were measured from 1983 using immunochemical methods and after 1985 using independently designed methods. The results showed deficient conditions in many Norwegian families. As warfarin reduces the blood concentration of protein C low warfarin doses and effective heparin treatment must be assured when patients are treated for protein C deficieny. In conclusion the importance of measurement of blood coagulation inhibitors (antithrombin protein C heparin cofactor II) in those susceptible to deep venous thrombosis is emphasized.