Severe prekallikrein (Fletcher factor) deficiency due to a compound heterozygosis (383Trp stop codon and Cys529Tyr)

We investigated a family with prekallikrein deficiency, using both standard coagulation tests and molecular biology techniques. The propositus was found to be a compound heterozygote for a Trp383 stop codon and a Cys529Tyr point mutation. The former mutation was located in exon 11, the latter in exon 14. The propositus inherited the first defect from his father and the second from his mother. Both parents had slightly low prekallikrein levels, but the combination of the two genetic defects produced a phenotype characterized by an extremely low prekallikrein activity and antigen. The propositus' plasma showed a progressive reduction in APTT when incubated for a long time. Conversely, plasma deficient in factor XII, factor XI or high molecular weight kininogen (HMWK) failed to show shortening of the APTT. No circulating anticoagulant was found because the patient's APTT was fully corrected by pooled nor-mal and factor XII-, factor XI- or HMWK deficient plasma. No associated abnormality was apparent in the propositus or his parents. As expected, no tendency for bleeding was noted even after tonsillectomy.     

Prekallikrein deficiency: the characteristic normalization of the severely prolonged aPTT following increased preincubation time is due to autoactivation of factor XII

Hereditary plasma prekallikrein (PK) deficiency was diagnosed in a 71-year-old man with an 8-year history of osteomyelofibrosis. PK deficiency was suspected in view of a severely prolonged activated partial thromboplastin time (aPTT) that nearly normalized following prolonged preincubation (10 min) of patient plasma with kaolin–inosithin reagent. Hereditary PK deficiency was demonstrated by very low PK values in the propositus (PK clotting activity 5%, PK amidolytic activity 5%, PK antigen 2% of normal plasma, respectively) and half normal PK values in his children.

Normalization of a severely increased aPTT (>120 s) after prolonged preincubation with aPTT reagent occurred in plasma deficient in PK but not in plasma deficient in factor XII (FXII), high-molecular-weight kininogen (HK), factor XI (FXI), factor IX, factor VIII, Passovoy trait plasma or plasma containing lupus anticoagulant. Autoactivation of FXII in PK-deficient plasma in the presence of kaolin paralleled the normalization of aPTT. Addition of OT-2, a monoclonal antibody inhibiting activated FXII, prevented the normalization of aPTT. We conclude that the normalization of a severely prolonged aPTT upon increased preincubation time (PIT), characteristic of PK deficiency, is due to FXII autoactivation.     

Congenital protein C deficiency and myocardial infarction:concomitant factor VII hyperactivity may play a role in the onset of arterial thrombosis.

A 29-year-old man with congenital protein C deficiency and acute myocardial infarction is reported. Four hours after the onset of chest pain, he was treated intravenously with tissue-type plasminogen activator. Subsequent coronary angiography revealed only slight stenosis of the left anterior descending coronary artery without any atherosclerosis. The propositus, his brother, and his mother, showed low levels of both protein C activity and antigen, while plasma thrombomodulin levels were normal. His grandfather had died from acute myocardial infarction at 38 years of age. We investigated several other risk factors for arterial thrombosis, including factor VII, fibrinogen, heparin cofactor II, lipoprotein (a), and anticardiolipin antibodies. No other haemostatic abnormalities apart from factor VII hyperactivity were detected in this family. To study the effects of protein C and factor VII on procoagulant activity, prothrombin time was measured after the addition of activated protein C and factor VII to protein C-deficient plasma. The prothrombin time ratio decreased along with an increase in the factor VII level. It also decreased with a decrease in the activated protein C level. These findings indicated that the procoagulant activity of factor VII was enhanced by low protein C levels, suggesting that concomitant factor VII hyperactivity may cause acute myocardial infarction in patients with protein C deficiency.     

Homozygous variant of antithrombin III: AT III Fontainebleau

A qualitative defect of antithrombin III (AT III) was demonstrated in four members of a large Tunisian family by the discrepancy between a normal amount of antigen and decreased or absent heparin cofactor activity. The propositus, a 3-year-old girl, died from massive intracardiac thrombosis despite oral anticoagulant therapy. Heparin cofactor activity measured in the presence of thrombin or F. Xa was undetectable in her plasma. Anti-F. Xa activity was also absent when using low molecular weight heparin or a synthetic pentasaccharide, representing the binding site to AT III. The lack of affinity of the propositus AT III for heparin was demonstrated by two-dimensional immunoelectrophoresis and chromatography on heparin-Sepharose. The parents, first cousins, and the sister of the propositus also demonstrated a qualitative abnormality of AT III, with levels of heparin cofactor activity close to 50% of the normal range. Our data support the view that the abnormal protein was present at the heterozygous state in the parents and sister and at the homozygous state in the propositus. None of the affected family members had thrombotic episodes, except for the propositus. The name of AT III Fontainebleau is proposed for this variant.     

Investigation of a congenital abnormal plasminogen, Frankfurt I, and its relationship to thrombosis

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who has recurrent deep vein thrombosis. Clinical laboratory data showed normal hemostasis test results. Since plasma plasmin generation rates gave low values, the fibrinolytic system was analyzed for a possible fibrinolytic system defect. Functional and antigen plasminogen concentrations both in the plasma and with the isolated, purified plasminogen showed that only 49% of the antigen concentration had potential functional active sites. Also, a reduced antigen concentration was found in both the propositus, and his mother (46% active sites). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified Frankfurt I plasminogen showed a normal native Glu-plasminogen band. Crossed-immunoelectrophoresis revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode, i.e., was, as a whole, more negatively charged. Isoelectric focusing followed by zymography on a agarose-fibrin plate proved this observation, but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin led to the conclusion that most of the functional abnormality is related to absence of active sites in half of the molecules. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. After reduction, the plasmin was completely converted to the typical two plasmin chains, A and B.     

Beta 2-glycoprotein I deficiency and the risk of thrombosis.

beta 2-glycoprotein I (beta 2-GP I) is a plasma protein with a high affinity for negatively charged surfaces. In vitro this protein shows a variety of anticoagulant properties (inhibition of contact activation and platelet dependent prothrombinase activity). Therefore we studied the possibility that a hereditary beta 2-GP I deficiency is a risk factor for (familial) thrombophilia. Plasma beta 2-GP I levels were measured in healthy volunteers and four different groups of patients with (familial) thrombophilia. In these 5 groups the prevalence of beta 2-GP I deficiency (i.e. beta 2-GP I antigen less than 77%) was found to be very similar (6.8-12.5%) and statistically not significantly different. This observation suggests that beta 2-GP I deficiency in itself is not a risk factor for thrombosis. One thrombophilic patient was found to be homozygous deficient of beta 2-GP I. The transmission of the defect in his family followed autosomal inheritance. One of his brothers was also homozygous deficient and at the age of 35 years still free of thromboembolic complications. The possibility that beta 2-GP I deficiency could be an additional risk factor for the development of thrombophilia in families with protein C deficiency was evaluated in a panel of 70 unrelated patients with clinically dominant protein C deficiency. The prevalence of beta 2-GP I deficiency in this group of patients (12.8%) was very similar to that in other groups of normals and patients. Moreover, there was no difference in the frequency of beta 2-GP I deficiency in symptomatic and asymptomatic protein C deficient patients.     

Variations of protein C in uremic hemodialysed patients

Protein C has been measured by three different assays (antigenic, amidolytic and chronometric) in 27 end-stage renal insufficient patients before and after hemodialysis. Protein C levels have been compared with other coagulation inhibitors (antithrombin III, protein S) and fibrinolytic parameters. Baseline anticoagulant activity of protein C has been found impaired in eight cases whereas other inhibitors were normal. In four cases, both anticoagulant and antigenic levels were low. In one case, amidolytic method could also found a low activity. Hemodialysis leads to an increase of protein C activity and antigen level. Heparinemia after hemodialysis does not interfere with the chronometric measurement of protein C anticoagulant activity. Total protein level, hematocrit, protein S and antithrombin III are also elevated after hemodialysis. Baseline fibrinolytic parameters are normal and remain unchanged after hemodialysis. The clinical relevance of such modifications is discussed.     

Is the carboxyl-terminus of dystrophin required for membrane association? A novel,severe case of duchenne muscular dystrophy

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by the deficiency of a component of the muscle fiber membrane cytoskeleton called dystrophin. Becker muscular dystrophy, a clinically milder disorder, results from dystrophin abnormalities rather than deficiency. We identified the first patient who is clearly an exception to these established clinical and biochemical correlates. The patient described clinically had particularly severe Duchenne dystrophy. Biochemically, his muscle contained substantial amounts of abnormal dystrophin (Becker-like). Characterization of the dystrophin protein and gene revealed a unique intragenic gene deletion resulting in a dystrophin protein missing the carboxyl-terminal domain. This patient's dystrophin seemed to have a deleterious "dominant" effect on his muscle: The presence of this abnormal protein was more damaging to the myofibers than the absence of dystrophin would have been. This patient challenges the current hypothesis that dystrophin associates with the plasma membrane solely via its carboxyl-terminus, yet supports the hypothesis that an intact carboxyl-terminus is crucial for correct dystrophin function.     

A functional assay of protein C in human plasma

A functional assay for protein C in plasma is described in which barium eluates of plasma are incubated with bovine thrombin and rabbit thrombomodulin to activate protein C. The activated protein C solution is added to an activated partial thromboplastin time (APTT) system containing normal plasma and an APTT reagent (Dade ActinR). The prolongation of coagulation time after recalcification in this system is taken as a measure of the anticoagulant activity of protein C. When expressed as per cent of the value in pooled normal plasma, the results obtained by this method in 34 normal controls and in 3 untreated patients with protein C deficiency were very similar to those obtained by radioimmunoassay of protein C. In 2 patients with protein C deficiency and 23 patients without, all on dicoumarol or warfarin treatment, the anticoagulant activity of protein C was less than its antigen concentration. The day to day analytical coefficient of variation (SD/mean) was 12% at the 100% level (n = 12), and 10% at the 25% level (n = 12).     

“In vitro” fibrinolytic activity of a new low molecular weight heparan sulfate

The fibrinolytic activity, effect on the fibrinolytic activity of plasmin, anticoagulant activity and anti platelet aggregation activity of a low molecular weight heparan sulfate ( LMW HS Bal ) were investigated “in vitro” on blood plasma obtained from rats and rabbits. LMW HS Bal at concentrations as low as 0.25 – 2 μg/ml prevented thrombin-induced platelet aggregation. At concentrations 25 to 50 times larger it showed no significant anticoagulant activity but a marked fibrinolytic effect. At still larger concentrations LMW HS Bal also potentiated the fibrinolytic activity of plasmin. Conversely, unfractionated heparan sulfate (UHS) at concentrations of 25 to 50 μg/ml, only showed anticoagulant activity with no fibrinolytic activity.     

Atypical CADASIL phenotypes and pathological findings in two new French families]

Atypical phenotypes of CADASIL and corresponding anatomical data in two cases are described in 6 members of 2 new French families. In the first family, 4 cases in the same kindred were probably affected, two of them with a predominant psychiatric presentation, two others with dementia and a pseudo-bulbar syndrome of progressive evolution. No history of migraine or ischemic event were documented in any. In the propositus, the diagnosis was documented by skin biopsy, Notch 3 gene mutation and autopsy after the patient had died when 67 years old, 8 years after onset. Brain examination showed a widespread leukoencephalopathy with subcortical infarcts. Characteristic granular lesions of the small arteries of the brain and other organs were observed. In the second family, two cases are reported. One patient died when 63 years old after a subacute evolution mimicking intracranial hypertension. The anatomical diagnosis was retrospectively proven typical of CADASIL with Notch 3 immunostaining of arterial smooth muscle cells. The other case had a progressive evolution over 20 years of limb paresthesia with a mild spasticity diagnosed as a progressive form of multiple sclerosis. It was followed by a pseudo-bulbar syndrome and a mild subcortical dementia without acute ischemic attack. The diagnosis was confirmed by skin biopsy and mutation of the Notch 3 gene. This report illustrates     

Dysfunctional activated protein C (PC Cádiz) in a patient with thrombotic disease

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.     

Familial amyotrophic lateral sclerosis

Familial occurrence of neuromuscular disease similar to sporadic amyotrophic lateral sclerosis has been reported from several countries.
A Norwegian family with muscular wasting in men and women of three generations is described. The propositus and his father's sister were examined, as well as a second cousin of the propositus. The disease was characterized by late onset, predominantly upper limb peripheral pareses, and "pyramidal" signs in the lower extremities. Although peripheral neuromuscular affection in the lower limbs tended to be subclinical, chonchotome biopsies from the tibialis anterior muscle showed neurogenic atrophy in all three cases.     

Congenital deficiency of plasminogen and its relationship to venous thrombosis

In a patient with deep venous thrombosis, plasma concentrations of coagulant and inhibitor proteins were normal except for moderate deficiency of plasminogen. Family studies revealed a similar deficiency in the mother and sister of the propositus. Evaluation of purified plasminogen demonstrated that it functioned normally. The patient represents our only example of plasminogen deficiency in 435 German individuals evaluated with a history of thromboembolism.     

Hereditary antithrombin III deficiency with a superior sagittal sinus thrombosis: evidence for a possible mutation starting in the mother of the propositus

A patient with a superior sagittal sinus thrombosis (SSST) which occurred during the postpartum period is reported. Coagulation studies revealed persistently decreased antithrombin III (AT III) activity. Studies of AT III activity in her family revealed a hereditary AT III deficiency. Since AT III activity was normal in the grandparents of the propositus, the disorder in this family appears to be due to a spontaneous mutation occurring in the mother of the propositus.     

Beh?et syndrome associated with protein S deficiency.

Beh?et syndrome is a multisystem disorder characterized by ocular, mucocutaneous, articular, gastrointestinal and neurologic abnormalities. We report here an unusual case of Beh?et syndrome, characterized by the importance of the thrombotic events (7 phlebitis of both legs and resection of two toes). Additional manifestations of the Beh?et syndrome occurred only 10 years after the first thrombotic episode. The oldest daughter of the propositus and his brother suffered also from thrombophlebitis; this familial history of thrombosis led to the performance of a haemostatic study. A congenital protein S deficiency was found in the propositus and in three of his children. Normal protein S levels were found in nine unrelated patients with Beh?et syndrome. Thus this observation suggests that, when thrombotic manifestations are the first and major symptom of Beh?et syndrome, an additional cause of thrombosis has to be investigated.     

Co-administration of low molecular weight heparin enhances the profibrinolytic effect of warfarin through different mechanisms

Background and Objective

Treatment with vitamin K antagonists (VKA) reduces fibrinolytic resistance through the inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor (TAFI). Because low-molecular weight heparin (LMWH) is co-administered with VKA during initiation of anticoagulant treatment, we evaluated the effect of dual anticoagulation on fibrinolytic resistance.

Patients and Methods

Two groups of patients were studied: 1) patients on stable warfarin; 2) patients starting oral anticoagulant therapy, who were evaluated during dual anticoagulation and after enoxaparin withdrawal. Only samples with an INR between 2 and 3 were compared. The resistance of clots to t-PA-induced fibrinolysis was evaluated in blood and plasma by thromboelastography (TEG) and turbidimetry, respectively.

Results

In patients on dual anticoagulation, blood fibrinolysis time (TEG) was significantly shorter than in patients on warfarin alone and significantly correlated with LMWH level. The profibrinolytic effect was partly ascribable to a reduction of thrombin-dependent TAFI activation: 1) thrombin and TAFIa generation were significantly reduced by dual anticoagulation; 2) the addition of enoxaparin to warfarin-blood reduced TAFI-mediated fibrinolysis inhibition. Patients on dual anticoagulation also displayed a reduction in clot strength, a phenomenon known to reduce fibrinolytic resistance. The profibrinolytic effect of LMWH co-administration was not seen in plasma, likely because TAFIa generation was below the threshold required to inhibit fibrinolysis.

Conclusions

Co-administration of LMWH in patients under VKA reduces the fibrinolytic resistance of blood clots via TAFI-dependent and TAFI-independent mechanisms. Further studies are warranted to assess the clinical implications of these findings.     

Hereditary homozygous heparin cofactor II deficiency and the risk of developing venous thrombosis.

Heparin cofactor II (HCII) is a specific inhibitor of thrombin in the presence of heparin or dermatan sulphate. Although there have been reports on families in which a heterozygous HCII deficiency is associated with thromboembolic events, several epidemiological studies revealed that heterozygous HCII deficiency is as prevalent among healthy subjects as it is among patients with deep venous thrombosis (DVT). It is therefore not yet clear whether HCII is or is not a thrombotic risk factor. We analyze and describe in an extended family the biochemical and genetic thrombophilic risk factors and evaluate the potential thrombotic risk involved in homozygous and heterozygous HCII deficiency, either alone or associated with other thrombotic or circumstantial risk factors. The propositus has had three episodes of DVT and a pulmonary embolism. During the first episode of DVT the patient was diagnosed as having AT deficiency. Later, a functional and antigenic HCII deficiency, compatible with the homozygous form, was detected. The family study shows that both the propositus and her sister have homozygous HCII deficiency and that 12 of the 27 family members have heterozygous HCII deficiency. This is possibly the first case report on a homozygous phenotype for the HCII deficiency with. in addition, partial AT deficiency. The propositus has suffered several thrombotic events, unlike the other 12 family members with heterozygous HCII deficiency and her sister, who is also homozygous for this disorder. We suggest that HCII deficiency may play a limited in vivo role as a thrombotic risk factor unless associated with AT deficiency or another congenital thrombotic risk factor.     

A family with probable autosomal dominant bulbospinal muscular atrophy with gynecomastia]

We reported a 52-year-old man and his family with bulbospinal muscle atrophy (BSMA) and gynecomastia. The propositus presented with the clinical picture of late onset progressive bulbospinal muscular atrophy including postural tremor, general hyporeflexia, mild maturity onset diabetes, gynecomastia and sexual impotence. One of his brother and his two sons had gynecomastia. His elder son had ocular movement abnormality, associated movement of facial muscle and finger tremor. One of his brothers showed tongue fasciculation without gynecomastia. None of members examined had abnormal expansion of CAG repeats in the androgen receptor gene. We speculate that this family has a new clinical entity characterized by bulbospinal muscular atrophy with an autosomal dominant inheritance.     

Juvenile Sandhoff disease with local panatrophy--a case report

A case of juvenile Sandhoff disease with Gowers' local panatrophy was reported. A mentally retarded 39 year-old man had been noticed to have localized skin atrophy on the right leg since 7 years of age, and was also aware of the atrophy of his right leg since his junior high school age. His parents were first cousins. He had multiple localized atrophic skin lesions on the right side of his nape, the right side of lower back, and the right thigh. These skin lesions did not show any signs of sclerosis. Although he was mentally retarded (IQ=44), the neurological examination was normal including funduscopy. Histological examination of the atrophic skin le led that the epidermal basal layer was hyperpigmented and that the dermis and subcutaneous fatty tissue were extremely atrophic. The nerves meandered across the dermis and subcutaneous tissue, and had membranous lipid storage cytosomes in the axon and Schwann cells. Biochemical studies revealed marked deficiency fo leukocyte hexosaminidase activity. Total hexosaminidase activity was decreased to 18% of normal controls, and hexosaminidase B activity was completely deficient. Other lysosomal enzymes in leukocytes were normal in activities. Both parent showed intermediate levels of leukocyte hexosaminidase A and B activities. Electron microscopy analysis of a rectal biopsy specimen showed neuronal accumulation of dense osmophilic deposits, as well as membranous cytoplasmic bodies in Meissner's plexus. On the basis of the patient's age at onset and the above findings, the patient was diagnosed to have juvenile Sandhoff disease. The local panatrophy observed in the present case could be the result of the accumulation of GM2-ganglioside in autonomic nervous system, and may well be a new clinical feature of GM2-gangliosidosis.