Section 10: Nutrients and their functions Vitamin K and blood clot formation 01/27/06

Vitamin K The RDA for vitamin K is 80  g for men and 65  g for women (age 18-50). Fat soluble. Common to many foods, and produced by intestinal bacteria. Required for the carboxylation of glutamate side chains of several proteins, especially those involved in blood coagulation. The RDA for vitamin K is 80  g for men and 65  g for women (age 18-50). Fat soluble. Common to many foods, and produced by intestinal bacteria. Required for the carboxylation of glutamate side chains of several proteins, especially those involved in blood coagulation. 1

Hemostasis There are three immediate responses to blood vessel trauma. –Vascular spasm –platelet aggregation ( and release of vasoconstrictors: thromboxane A2, serotonin, epinephrine ). –Fibrin clot formation (requires vitamin K) The mesh of fibrin clots and aggregated platelets plug the opening. Clot formation occurs as a result of a cascade of proteolytic reactions. There are three immediate responses to blood vessel trauma. –Vascular spasm –platelet aggregation ( and release of vasoconstrictors: thromboxane A2, serotonin, epinephrine ). –Fibrin clot formation (requires vitamin K) The mesh of fibrin clots and aggregated platelets plug the opening. Clot formation occurs as a result of a cascade of proteolytic reactions. 2

Clot Formation Soluble fibrinogen is circulating in the blood. Thrombin is a proteolytic enzyme that hydrolyzes fibrinogen to fibrin monomer and small fibrinopeptides A and B. Fibrin is insoluble and aggregates. Thrombin also activates transglutaminase (factor XIIIa) which cross-links fibrin monomers, making the clot mechanically strong. Soluble fibrinogen is circulating in the blood. Thrombin is a proteolytic enzyme that hydrolyzes fibrinogen to fibrin monomer and small fibrinopeptides A and B. Fibrin is insoluble and aggregates. Thrombin also activates transglutaminase (factor XIIIa) which cross-links fibrin monomers, making the clot mechanically strong. 3

Fibrin Aggregation Fibrin monomers spontaneously aggregate into long rod-shaped structures. The structure is mechanically weak. Fibrin monomers spontaneously aggregate into long rod-shaped structures. The structure is mechanically weak. 4

Crosslinking Reaction Peptide bonds connect glutamine and lysine residues on different fibrin monomers in the fibrin aggregate. Catalyzed by transglutaminase (XIIIa). This greatly strengthens the aggregate. Peptide bonds connect glutamine and lysine residues on different fibrin monomers in the fibrin aggregate. Catalyzed by transglutaminase (XIIIa). This greatly strengthens the aggregate. p287 5

Fibrinogen Fig Fibrinogen is a 340 kD complex of 2 A , 2 B  and 2  subunits. Thrombin removes A and B proteolytically from the amino ends of the A  and B  subunits, producing fibrin monomer and 4 small fragments (fibrinopeptides). Fibrinogen is a 340 kD complex of 2 A , 2 B  and 2  subunits. Thrombin removes A and B proteolytically from the amino ends of the A  and B  subunits, producing fibrin monomer and 4 small fragments (fibrinopeptides). 6

Fibrin monomer to fibrin clot Fig Fibrin monomer is insoluble and polymerizes. The globular carboxy ends of  bind the thrombin-modified amino ends (“knobs”) of red A  subunits. The globular carboxy ends of blue B  bind the amino ends of neighboing B  to form cable structures (not shown). Fibrin monomer is insoluble and polymerizes. The globular carboxy ends of  bind the thrombin-modified amino ends (“knobs”) of red A  subunits. The globular carboxy ends of blue B  bind the amino ends of neighboing B  to form cable structures (not shown). 7

Fig Clot Cascade A series of enzyme activations by proteolytic cleavage lead to fibrin clot production. Fibrin is active to aggregate. It is not a proteolytic enzyme. Factor VIII is the anti-hemophilia factor. VIIIa is more active than VIII. A series of enzyme activations by proteolytic cleavage lead to fibrin clot production. Fibrin is active to aggregate. It is not a proteolytic enzyme. Factor VIII is the anti-hemophilia factor. VIIIa is more active than VIII. 88

Intrinsic and Extrinsic Pathways INTRINSIC PATHWAY. A foreign surface (collagen) activates high-molecular-weight kininogen, prekallikrein. Kallikrein activates factor XII. XIIa produces more kallikrein, in an autoactivation cycle. EXTRINSIC PATHWAY. Trauma activates factor VII and releases tissue factor (III), a lipoprotein from blood vessels. Tremendous amplification. Each step is proteolytic conversion of proteolytic enzyme to its active form (subscript ‘a’). VIIIa, tissue factor, and fibrin are not enzymes. INTRINSIC PATHWAY. A foreign surface (collagen) activates high-molecular-weight kininogen, prekallikrein. Kallikrein activates factor XII. XIIa produces more kallikrein, in an autoactivation cycle. EXTRINSIC PATHWAY. Trauma activates factor VII and releases tissue factor (III), a lipoprotein from blood vessels. Tremendous amplification. Each step is proteolytic conversion of proteolytic enzyme to its active form (subscript ‘a’). VIIIa, tissue factor, and fibrin are not enzymes. 99

Intrinsic and Extrinsic Pathways Converge to Produce Thrombin Both pathways are required for adequate levels of Xa to produce sufficient amounts of thrombin. PF3 is platelet factor 3. Both pathways are required for adequate levels of Xa to produce sufficient amounts of thrombin. PF3 is platelet factor 3. 10

Prothrombin Structure Xa cleaves prothrombin at two sites to produce thrombin, a serine protease. Gla and kringle domains keep prothrombin inactive. Ten glu residues at the amino end of the Gla domain are carboxylated. The  -carboxyglutamates bind Ca 2+ and are required for prothrombin to have the correct conformation to be a substrate for Xa, and they anchor it to platelet membranes at trauma sites. Xa cleaves prothrombin at two sites to produce thrombin, a serine protease. Gla and kringle domains keep prothrombin inactive. Ten glu residues at the amino end of the Gla domain are carboxylated. The  -carboxyglutamates bind Ca 2+ and are required for prothrombin to have the correct conformation to be a substrate for Xa, and they anchor it to platelet membranes at trauma sites. Fig Fig Prothrombin 11

Carboxylation of Glutamate Glutamate sidechains are converted to  -carboxyglutamate in factors VII, IX, X and prothrombin. The two carboxyls of  -carboxyglutamate chelate Ca 2+. This is an example of post-translational modification. Glutamate sidechains are converted to  -carboxyglutamate in factors VII, IX, X and prothrombin. The two carboxyls of  -carboxyglutamate chelate Ca 2+. This is an example of post-translational modification. 12

Vitamin K Reaction Cycle Vitamin K is a cofactor for glutamate carboxylation reaction. The reductases use NADPH as an e - source. Vitamin K is a cofactor for glutamate carboxylation reaction. The reductases use NADPH as an e - source. 13

Thrombin Inactivation Clot formation and degradation are under close control. The protein antithrombin III binds to thrombin, is proteolyzed, and irreversibly inactivates the thrombin (not shown). The polysaccharide heparin enhances the binding of antithrombin III to thrombin. Clot formation and degradation are under close control. The protein antithrombin III binds to thrombin, is proteolyzed, and irreversibly inactivates the thrombin (not shown). The polysaccharide heparin enhances the binding of antithrombin III to thrombin. 14

Plasmin activation Plasminogen is bound in the fibrin- platelet mesh. TPA finger and growth domains bind fibrin and anchor TPA to a clot. TPA converts plasminogen to plasmin, which proteolytically degrades the clot. TPA is used therapeutically to reduce clot formation after a heart attack or stroke. Plasminogen is bound in the fibrin- platelet mesh. TPA finger and growth domains bind fibrin and anchor TPA to a clot. TPA converts plasminogen to plasmin, which proteolytically degrades the clot. TPA is used therapeutically to reduce clot formation after a heart attack or stroke. Stryer 4th 15

Fibronectin Fibronectin is a large multidomain glycoprotein present in connective tissue. At sites of trauma it helps to “coordinate” the repair process by binding several of the proteins involved. Fibronectin is a large multidomain glycoprotein present in connective tissue. At sites of trauma it helps to “coordinate” the repair process by binding several of the proteins involved. Fig Stryer 3rd 16

Vitamin K Analogs Menadiol is a water- soluble vitamin K analog. Warfarin is a potent inhibitor of clot formation, used after heart attacks, and also as rat poison (at higher doses). Menadiol is a water- soluble vitamin K analog. Warfarin is a potent inhibitor of clot formation, used after heart attacks, and also as rat poison (at higher doses). 17

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