INTRODUCTION

Hereditary Thrombophilia has become a major area of research in the field of hematology. Inherited causes of bleeding have been known for a long time but a lot of research is now going on in the inherited causes of thrombophilia. [1]Table I. 

Table 1: .Prothrombotic states 

Thromboembolism except that related to oral contraceptives was supposed to be a disease of the elderly. Now this disease is increasing being seen in the young patients who have no other risk factors. A hereditary cause must be ruled out in these patients.

Protein S (PS) deficiency is a rare cause of hereditary thrombophilia.  

PATHOPHYSIOLOGY

Inhibitors of the coagulation system

Checks and balances are present in every system of the body. The coagulation system has its own inhibitors. The inhibitors include protein C (PC), protein S(PS) and thrombomodulin.

Protein ‘C’ system

Protein C (PC) is the main vitamin K dependent protein that is active in inhibiting the coagulation system. It is synthesized in the liver. It is not active as such. It has to be activated. The activation is brought about by thrombin.

Thrombomodulin is another protein that combines with thrombin and greatly increases the rate of activation of PC. Thrombomodulin also depresses the thrombin’s potential of activation of other coagulation factors.(Fig A).

Figure A: Important coagulation reactions. Thick lines represent the fibrinolytic system. Thin lines represent the coagulation cascade.

Once the PC is activated it combines with PS and calcium ions. This complex (PC-PS-Ca++) has 10-20 times more power to degrade the active coagulation factors Va and VIIIa than PC alone. The cleavage of factor VIII takes place at a site that disables its binding capacity to combine with thrombin and factor IXa. The cleavage of factor Va takes place at a site that disables its binding capacity to combine with prothrombin and factor Xa.

The activated PC has its inhibitor known as plasminogen activator inhibitor type 3(PAI3).

Protein  ‘S’: Physiology

PS is a 70kDa glycoprotein that was discovered in 1984 in Seattle. It is a single chain protein and has 11 gamma carboxylated residues. It is a Vitamin K dependent protein. Its synthesis takes place in the megakaryocytes, vascular endothelium and the liver. It has a free and bound form. The free form comprises 40% of the total PS. The rest 60% is in combination of a binding protein the C4bBP. The bound form is inactive. It does not contribute to the coagulation reactions. So it is important to measure both the total and the free protein S in the serum. ‘Beta 2 glycoprotein I’ is a protein in the serum. Beta 2 glycoprotein I inhibits the binding of PS with C4bBP. It regulates the level of PS in the blood[2]. Like every other system PS also has its inhibitors. ‘Thrombin’ breaks down PS and halts its activity as a cofactor to PC. 

Serum protein ‘S’ Level

The total PS and the free PS levels can sometimes be normal but the PS activity can be low [3].

Total PS and free PS levels vary with age and sex. A study done on 3788 healthy volunteer blood donors in UK showed that men had a higher level of total and free protein S levels compared to women. In men there was a slight decrease in the level of free PS with increasing age but the total PS was normal. In women there was an increase in the level of total PS level with age. Oral contraceptives do cause a decrease in the level of the total protein S but the level of the free protein S is not affected [4]. 

Types of protein ‘s’ deficiency

Three types of PS deficiency are seen. The classification is based on functional and antigenic assay of the free, bound and total PS (A).

Type I PS deficiency

In type I PS deficiency the total, free and bound forms are all reduced.

Type II PS deficiency.

There is a normal antigenic level of the free and bound form of PS but the function is impaired.

Type III PS deficiency

There is a reduced level of free PS. The total PS is normal. In some families the Type I and II PS deficiency have been noted (A).  

ETIOLOGY OF PS DEFICIENCY

As far as the etiology of PS deficiency is concerned a number of genetic and environmental factors have been noted. [5]. (Table 2). 

Table. 2

Genetic mutations

The human PS gene is on chromosome 3 and contains two genes called alpha and beta. Transcription takes place at the alpha gene only (PG). Most of the mutations are of the missense type.

PROS I gene is supposed to be the gene responsible for the PS synthesis. Patients with PROS I gene defect and their first-degree relatives with similar defect were at a 5 times higher risk of thrombosis. [6].

Various mutations have been cited. New mutations in patients of deficiency of PS are being discovered [7], [8], [9], [10]. [11]. According to some authorities the PROS I gene mutation is of six types [12].

mRNA is the molecular messenger in the cell. Defective mRNA will result in production of abnormal proteins from the ribosomes. The mutation of PROS I gene causes production of a defective mRNA. It seems to be responsible for the reduced activity of Protein S [13].

Some authorities hold the view that there is poor co-relation between the genotype and the phenotype [14].

Families with multiple thrombophilic mutations have been seen [15]. A family with PS, PC deficiency and hereditary spherocytosis has been reported [16].

Acquired cases

Various factors have been found to depress the level of PS in the serum. 

Female hormones

Hormone therapy especially oral contraceptives are a well-known risk factor for thromboembolism in normal individuals. The pathogenesis has been traced to PS deficiency. Estrogen therapy causes a reversible PS deficiency in normal individuals during the treatment [17]. Oral contraceptives do cause a decrease in the level of the total protein S but the level of the free protein S is not affected [3]. If Oral contraceptives are given to patients with PS deficiency the risk for thrombophilia is increased further [18].

 During Pregnancy there is a gradual decrease in the level of PC and PS levels.  Therefore the measurement of PS and PC level is unreliable during pregnancy [19].

A recent study of the molecular structure of the PS revealed a sex hormone binding globulin (SHBG) like domain on the C terminal of protein S. [20]. 

Systemic lupus erythematosus(sle)

Systemic lupus erythmatosus is an autoimmune disease and there are various types of antibodies seen in this disease. Some studies found a low level of PS in the patients of SLE. Anti PS antibodies have been seen in patients of SLE. This results in a decreased level of PS in the serum [21]. Anti PS antibodies have been detected in 25.9% of patients of SLE. [22]. PS deficiency has also been seen in a patient with SLE with one of its complication i.e nephrotic syndrome [23]. There is a case report of a child with acute varicella infection who had a low level of PS and positive lupus anticoagulant in the serum [24]. 

Leukemias

A patient of chronic lymphocytic leukemia with thrombosis was found harboring inhibitors of PS in the serum .The antibodies disappeared after successful treatment [25]. 

Anti phospholipid syndrome

PS has a plasma inhibitor protein C4b. Beta 2 glycoprotein I inhibits the binding of PS with C4b. Beta 2 glycoprotein I is a protein that regulates the level of PS in the blood. In patients with antiphospholipid syndrome, there are anti beta 2 glycoprotein I antibodies that inhibit the activity of Beta 2 glycoprotein I in the blood. This will facilitate the binding of C4b with PS. Thus the activity of PS is decreased and there is thrombosis [2] 

Drugs

L Asparagenase is a drug used in Acute Lymphoblastic Leukemia. It causes a transient decrease in the level of PS [26]. PC that is closely related to PS as far as the function is concerned has been found to be depleted in patients taking Valproic acid. [27]

Inflammatory bowel disease

A decreased level of Protein S has been found in patients of inflammatory bowel disease. [28][29].

Prevalence

The prevalence of PS deficiency in our population is not known. The prevalence is different for various population groups. It also differs for the normal population and the thrombophilic patients.

In a study from western India on young (<45years) patients of DVT they found an incidence of 6.5% for PS deficiency. [30] In contrast to the previous study, a small study from India on juvenile patients with episodes of thromboembolism found no patients of PS deficiency [31].    

PS and PC deficiency are important thrombophilic factors in Chinese patients [32]. PS deficiency was the most frequent cause of thrombosis in a group of Chinese patients with hereditary thrombophilia. [33].

A Japanese study compared patients of DVT and normal controls for the presence of deficiencies of AT –III, PC and PS. The prevalence of PS in the normal subjects was 2.02% and in the patients of DVT it was 17.7%. [34]. According to another study the prevalence of PS deficiency in normal adult Japanese population was 1%. [35].  

In Mexican patients with thrombophilia a 2% prevalence of PS deficiency was found [36]. Frequency of PS deficiency was 2% in a family planning clinic in Auckland. [37].

In a study from Serbia on hereditary thrombophilia a 1.6% incidence of PS deficiency was found. Patients of arterial thrombosis were also included in the study.[38].

In a group of people from Netherlands with a symptomatic thrombophilia there was a 1.5% incidence of venous thromboembolism. Out of these PS had a share of 0.4% only [39].     

CLINICAL PRESENTATIONS

Acute Deep vein thrombosis (DVT) is diagnosed in 800,000 new patients every year(D). DVT is the most common presentation of PS deficiency. A typical patient of Deep vein thrombosis (DVT) presents with history of some risk factors like major surgery, recent delivery, oral contraceptives followed by pain and swelling of the calf. Sometimes the patients may present late with shortness of breath (pulmonary embolism). In young (< 45 years) patients with DVT and no obvious risk factors, 34% had a demonstrable cause for hereditary or acquired thrombophilia. [31].

A case report of a PS deficient young pregnant lady with cerebral vein thrombosis has been seen. [40]. Dural sinus thrombosis has been reported in-patient with PS deficiency [41]. PS deficiency is also a risk factor for arterial ischemic stroke and Sino venous thrombosis in children. [42].

Anterior spinal artery syndrome , idiopathic Pyle phlebitis, sclerosing peritonitis , thromboembolism of the mesenteric artery, aorta and right side of the heart , Budd Chiari syndrome , Moyamoya syndrome , and cases of Retinal vein occlusion have also been noted. [,43,44,45,46,47,48,].

Arterial thrombosis was not supposed to be associated with PS deficiency. Factors associated with genetic thrombophilia have not been found to predispose to arterial thrombosis [5]. But a recent study showed that patients of Acute Myocardial Infarction had a decreased level of PS [49]. A mass obstructing the ostium of the left coronary artery was found in a patient of PS deficiency [50].

In addition to thrombosis there has been a reported case of thalamic hemorrhage in a patient with abnormal protein S [51].

Necrosis of the skin with intake of coumarin is usually seen in protein C deficiency. It has also been reported with type II PS deficiency [52]. 

NEW TESTING TECHNIQUES FOR PS

A new technique of one-step, immuno-turbidimetric assay of free protein S (fPS) in plasma has been developed. The technique has the advantage of detecting free PS only and does not pick the serum protein S (PS)-C4b-binding protein complexes. It is fully automatic [53].

A new automated PS clotting assay has been developed which has been found superior to the antigenic method [54].

New investigations for detecting an episode of thromboembolism are becoming available. Coagulofibrinolytic immunochemical molecular markers such as thrombin-antithrombin III complex and D dimer may be useful in detecting such an episode. [55]. They will certainly be a great help to detect patients of Thromboembolism that go unnoticed.

MANAGEMENT

The management of an acute episode of thromboembolism is the same no matter what is the cause. The long-term management has no specific guide lines.

Commonly used anti coagulants drugs are Heparin, Warfarin, and thrombolytics.

Anticoagulants :-.Heparin therapy,Unfractionted heparin(UFH)

Heparin is a glycosaminoglycans with a molecular weight of 4 -30 kD. It binds to arginine and lysine sites on antithrombin III. This combination enhances the activity of AntithrombinIII. This complex is a serine protease inhibitor and inactivates factors IXa, XIa, Xa, XIIa and thrombin.

Low molecular weight heparins (LMWH)

Low molecular weight heparins are prepared by the chemical degradation of the heparins. The molecular weight of LMWH is between 2.5-15kD. The activity against factor Xa is 2-4 times more than the UFH. The bioavailabilty of LMWH is 100% compared to 50% for UFH. They do not require any monitoring. 

Warfarin.

The optimal activity of the Vitamin K dependent proteins (factor II, VII, IX, X, PS, PC) depends on the process of gamma carboxylation of glutamate residues on the N terminal part of these proteins. This gamma carboxylation needs a catalyst, KH2 (reduced form of Vitamin K). So an oxidized Vitamin K is formed during this reaction. It has to be recycled in to a reduced from by the vitamin K epoxide reductase enzyme. Oral anticoagulants interfere with this enzyme and inhibit the recycling of Vitamin K.

The monitoring of the anticoagulant activity of Warfarin is by means of the Prothrombin time (PT) and its standardized ratio the International normalization ratio (INR). The target INR for most indications should be 2.5+-0.5. In case of patients with a mechanical prosthetic valve, recurrent venous thrombosis event while on Warfarin and life threatening thrombosis with antiphospholipid syndrome the desired INR is 3.5(A).

One important consideration in protein S deficient patients is the Vitamin K dependent synthesis of this protein. Warfarin is the only mode of prolonged anti coagulation. Warfarin acts by Vitamin K antagonism. Inhibition of Vitamin K on one hand depletes Factor II, VII, IX and X but also depresses the Protein S level. Theoretically the achievement of adequate anti coagulation in these patients should be difficult.

Thrombolytic drugs

Thrombolytic drugs include streptokinase, urokinase and tissue plasminogen activator (tPA). These drugs dissolve the clot if given early. Thrombolytic therapy is indicated in acute massive pulmonary embolism. In cases of DVT it is reserved for patients of massive proximal limb vein thrombosis and impending venous gangrene (A).

Heparin can be given as a continuous i/v infusion initially followed by intermittent s/c injections. An intravenous bolus of 7500-10,000 units followed by a continuous infusion of 1000-1500 unit/ hours is to be given (B). The anti coagulation effect depends on the dose. Heparin can also be given as intermittent subcutaneous injections (C). A daily dose of 30,000-35,000 units per day is supposed to produce a desirable anticoagulant response and has to be titrated against the APTT values(A). An APTT of 1.5-2.5 times the normal range is desirable. The LMWH can be given once or twice daily s/c according to the body weight. They do not require any monitoring. This is because they have predictable pharmacokinetics. The LMWH produce a desirable anticoagulation after the first dose compared to UFH that may need some dose adjustments over several doses(A).

Prophylactic Heparin is definitely indicated for the PS deficiency patients.  Hip surgery, prolonged immobilization, oral contraceptives, pregnancy etc that are risk factors for the   normal population are also risk factors for these thrombophilics. UFH can be given at a dose of 5000 units 8 hourly or 12 hourly. Several studies have established the efficacy of LMWH as a prophylactic heparin(A).

There are no clear-cut guidelines regarding the long-term management with Warfarin and the duration of anticoagulation for the PS deficient patients. Duration of anticoagulation depends on a number of factors e.g. whether the DVT was spontaneous or secondary to some cause, age of the patient, first or second episode of DVT and the time that has elapsed from the occurrence of DVT [56]. The duration of anticoagulation also depends on the presence and reversibility of the risk factors. Patients with reversible risk factors like surgery may need a three month period of anticoagulation. Idiopathic thrombosis and persistent risk factors like cancer, PS and PC deficiency may need a longer period like 6 months or longer of anticoagulation. The decision varies from patient to patient. [57].

Protein S deficiency predisposes a very small number of those affected to life-threatening thromboses and emboli, for which they are required to take lifelong prophylactic anticoagulation.[58] 

CONCLUSION 

Patients who have thromboembolic disease at a young age with no provoking event or who have a positive family history or whose thrombosis involves an unusual site should be investigated for hereditary thrombophilia.[59].  

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