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A Practical Guide to Haemostasis


Snake Venoms and their use in Haemostasis

Introduction

Snake venoms have evolved to subdue and digest their prey or to repel their enemies. The venoms consist of a vast number of components a number of which have been employed in laboratory tests and in some cases as therapeutic agents.

Snake venoms can be classified on the the basis of their effects on haemostasis:

Snake Venoms Components that affect Haemostasis
Procoagulant Clotting of Fibrinogen
Prothrombin activation to Thrombin
Factor X activation
Factor V Activation
Anticoagulant Protein C activation
Antithrombin activation
Denaturation of Factor V
Thrombin inhibitors
Fibrinolytic Degradation of Fibrinogen
Activation of Plasminogen to Plasmin
Platelet Function Platelet aggregation
Inhibition of aggregation
Vessel wall Haemorrhagins


The following list is not comprehensive but outlines some of the more commonly used venoms and their place in the Laboratory.

Snake venoms in the Laboratory

Taxonomic Name Common Name Location Active component Action
Thrombin-Like Enzymes
Bothrops atrox Common Lancehead South America Batroboxin also known as Reptilase 1. Cleaves Fibrinogen at Arg16-Gly17 releasing Fibrinopeptide A (FpA) and generating Fibrin.
Calloselasma rhodostoma formerly Agkistrodon rhodostoma Malayan Pit Viper Southeast Asia Ancrod 1. Cleaves Fibrinogen and is a powerful Defibrinating agent.
2. Factor XIII
Agkistrodon c. contortrix Copperhead snake Central/Eastern North America Protac 1. Cleaves Fibrinogen beta chain
2. Protein C activator
Prothrombin Activators
Pseudonaja textilis Eastern or Australian Brown snake Eastern and central Australia and southern New Guinea Textarin Prothrombin activation
Echis carinatus
Saw-scaled or Carpet Viper Middle East and Central Asia, and especially the Indian subcontinent Ecarin Prothrombin activation
Oxyuranus scutellatus Coastal or Common Taipan Coastal regions of northern and Eastern Australia and the island of New Guinea Taipan Prothrombin activation
Factor V, X Activators
Daboia russelii  Russell Viper Asia throughout the Indian subcontinent, much of Southeast Asia, southern China and Taiwan Russell Viper  Venom Factor V and Factor X activation
Protein C and APC Resistance
Agkistrodon c. contortrix Copperhead snake Central/Eastern North America Protac Protein C activator
Von Willebrand Factor
Bothrops jararaca Brazilian pit viper Southern Brazil, Paraguay, and northern Argentina Botrocetin Binds to the GpIb receptor on the surface of platelets
Fibrinolytic Enzymes
Crotalidae Pit Viper Eurasia and the Americas    
Viperidae Viperids Widely found    
Elapidae Elapids Endemic to tropical and sub-tropical regions around the World.    
Venom Proteins Acting on Vessel Walls
Crotalidae Pit Viper Eurasia and the Americas    
Viperidae Viperids Widely found    
Venom Proteins Acting on Vessel Walls
These can be divided into:
  a. C-type Lectins
  b. Disintegrins
  c. Thrombin-Like Enzymes
  d. 5' Nucleotidases
  e. L-Amino Acid Oxidases
  f. Phospholipase A2


Thrombin-Like Enzymes

Snake venoms that contain Thrombin-like enzymes have been isolated from a number of snakes The enzymes from these snakes are not inhibited by Heparin and can, therefore, be used to test plasma samples that contain Heparin and/or to remove Fibrinogen from samples that contain Fibrinogen.
Batroboxin also known as Reptilase, is isolated from the snake Bothrops atrox and forms the basis for a functional test of Fibrinogen - the Reptilase or Batroboxin Time that is very similar to the Thrombin Time but is unaffected by the presence of Heparin. A prolonged Reptilase time but a normal Thrombin Time usually indicates the presence of Heparin in the plasma sample. Most of the these Thrombin-like enzymes do not activate Factor XIII or the receptors on platelets.

Thrombin-Like Enzymes [TLE] isolated from snake venoms can be classified into 3 groups based upon the release of Fibrinopeptides A and B when Fibrinogen is activated - Venombin  A, Venombin B and Venombin AB - see References for further information.

Prothrombin Activators

Snake venoms that activate Prothrombin [Factor II] include: Echinus carinatus, Pseudonaja textilis and Oxyuranus s. scutellatus. These enzymes will cleave the functionally abnormal Prothrombin present in patients on a Vitamin K antagonist such as Warfarin, in addition to normal Prothrombin.  This property has been used to form the basis for a test that screens for PIVKAs - Proteins Induced by Vitamin K Antagonists.

The Prothrombin activators are classified into 4 groups:
1. Group A - Prothrombin activators that are metalloproteinases and activate Prothrombin without any requirement for cofactor e.g. Ca2+, Phospholipid or Factor Va.
2. Group B - Prothrombin activators that are Ca2-dependent.
3. Group C - Prothrombin activators  requiring Ca2 and negatively charged Phospholipid.
4. Group D -   Prothrombin activators that are serine proteases and are strongly dependent on Ca2+negatively charged Phospholipid and FVa.

Factor V Activators

The venom from Russell's viper - Daboia russelli contains a serine protease that cleaves Factor V at Arg145 and this is independent of Factors VII, VIII, IX, XI, and XII and so the test is unaffected by deficiencies or inhibitors of these proteins.
This property forms the basis for a Factor V assay although this it is not a widely performed test.
Russell viper venom has also be used as the basis for a test to screen for the Factor V Leiden abnormality in whole blood samples.

Factor VII and Factor X Activators

The venom from Russell's viper - Daboia russelli also contains a potent activator of Factor X. This property has formed the basis for a number of tests:
a. RVV Factor X Assay.
One of several methods for assaying Factor X.
b. Stypven Time:
The Stypven Time is another name for the Russell Viper Venom clotting time.  Russell Viper venom is a direct activator of Factor X and so the Stypven time is normal in Factor VII deficiency but abnormal in Factor V, II and most cases of Factor X deficiency.
c. Lupus Anticoagulant: The Dilute Russell Viper Venom Time [dRVVT]
In individuals with a Lupus Anticoagulant [LA], the Lupus Anticoagulant [antibody] binds to the Phospholipid inhibiting the action of the RVV and prolonging the clotting time ie the time to formation of a Fibrin Clot.
As the RVV directly activates Factor X, the test is unaffected by deficiencies of Factors XII, XI, IX or VIII. The dRVVT is frequently combined with a platelet neutralisation procedure to demonstrate the phospholipid specificity of the antibody i.e. the Lupus Anticoagulant.

Venoms of Value in Protein C Assays and APC Resistance

The venom from Agkistrodon c. contortrix - the Southern Copperhead snake contains a potent activator of Protein C - Protac. Protac is used in a number of tests including:
a. Functional Clotting-based and Chromogenic Protein C assays
b. Functional Protein S assays.
c. Global Protein C Assay - ProCŪ Assay.  This test was designed to rapidly screen for abnormalities in the Protein C-Protein S pathway including APC resistance.

Von Willebrand Factor

Botrocetin is a venom isolated from Bothrops jararaca. It binds to the GpIb receptor on the surface of platelets but at a site that is different from the binding or Ristocetin and has been used in von Willebrand Factor assays.

Venoms used in screening for a Lupus Anticoagulant

A number of snake venoms have been used to screen for the presence of a Lupus Anticoagulant.  These include:
a. The Taipan venom time employs a reagent isolated from the venom of the Taipan snake (Oxyuranus scutellatus) that directly activates Prothrombin in the presence of Phospholipid and calcium.
b. The Textarin Ratio: The Textarin:Ecarin ratio is a test for a Lupus Anticoagulant [LA] based on the differential dependence/requirement of these two snake venoms on Phospholipid to activate coagulation. Textarin, a protein fraction isolated from the venom of the Australian Eastern brown snake (Pseudonaja textilis) directly activates Prothrombin but requires Factor V, Calcium and Phospholipid to do so; whereas, Ecarin, a venom from the saw-scaled viper (Echis carinatus) activates Prothrombin to form Meizothrombin in the absence of Phospholipid. In the presence of a LA, the Textarin time is prolonged due to its phospholipid-dependence but the Ecarin time is not.
c. The Taipan:Ecarin ratio:  The Taipan:Ecarin ratio is very similar to the Textarin:Ecarin ratio.  The Prothrombin activator present in the venom of the Coastal Taipan (Oxyuranus scutellatus) activates Prothrombin in the presence of Calcium and Phospholipid to the intermediate Meizothrombin activating Fibrinogen and facilitating in vitro clot formation.
d. Dilute Russell Viper Venom Time [dRVVT]: see above.

Fibrinolytic Enzymes Isolated from Snake Venoms

A number of snake venoms have been shown to affect Fibrinolysis.  These Fibrinolytic enzymes can be divided into two groups:
A. Metalloproteinases and which degrade the Aα chain of Fibrinogen
B. Serine Proteases - these degrade the Bβ chain of Fibrinogen
.... although this specificity is not absolute.

A direct acting Plasminogen Activator is found in the venom of the Trimeresurus stejnegeri snake and Plasminogen activators have also been identified in the Lachesis muta muta and Agkistrodon halys snake venoms.

Venom Proteins acting on Vessel Walls

The Crotalidae and Viperidae venoms contain many metalloproteinases which degrade the blood vessel and extracellular matrix.  Based upon their domain structure, these metalloproteinases are divided into 4 major groups: P-I, P-II, P-III and P-IV.  See References for additional information.

Venom Proteins acting on Platelets

A number of proteins isolated from various snakes have been shown to have effects on platelets and platelet function.  These can be divided into:
a. C-type Lectins
b. Disintegrins
c. Thrombin-Like Enzymes - see previously
d. 5' Nucleotidases
e. L-Amino Acid Oxidases
f. Phospholipase A2

For more information - see References.